Methods and compositions for diagnosis and prognosis of renal injury and renal failure

ABSTRACT

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using assays that detect one or more of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and Insulin like growth factor-binding protein 6 as diagnostic and prognostic biomarker assays in renal injuries.

The present invention claims priority from U.S. Provisional Patent Application No. 62/160,519 filed May 12, 2015; from U.S. Provisional Patent Application No. 62/160,523 filed May 12, 2015; from U.S. Provisional Patent Application No. 62/160,526 filed May 12, 2015; and from U.S. Provisional Patent Application No. 62/160,531 filed May 12, 2015, each of which is hereby incorporated in its entirety including all tables, figures, and claims.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

The kidney is responsible for water and solute excretion from the body. Its functions include maintenance of acid-base balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function through injury and/or disease results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill, N.Y., pages 1741-1830, which are hereby incorporated by reference in their entirety. Renal disease and/or injury may be acute or chronic. Acute and chronic kidney disease are described as follows (from Current Medical Diagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, N.Y., pages 785-815, which are hereby incorporated by reference in their entirety): “Acute renal failure is worsening of renal function over hours to days, resulting in the retention of nitrogenous wastes (such as urea nitrogen) and creatinine in the blood. Retention of these substances is called azotemia. Chronic renal failure (chronic kidney disease) results from an abnormal loss of renal function over months to years”.

Acute renal failure (ARF, also known as acute kidney injury, or AKI) is an abrupt (typically detected within about 48 hours to 1 week) reduction in glomerular filtration. This loss of filtration capacity results in retention of nitrogenous (urea and creatinine) and non-nitrogenous waste products that are normally excreted by the kidney, a reduction in urine output, or both. It is reported that ARF complicates about 5% of hospital admissions, 4-15% of cardiopulmonary bypass surgeries, and up to 30% of intensive care admissions. ARF may be categorized as prerenal, intrinsic renal, or postrenal in causation. Intrinsic renal disease can be further divided into glomerular, tubular, interstitial, and vascular abnormalities. Major causes of ARF are described in the following table, which is adapted from the Merck Manual, 17^(th) ed., Chapter 222, and which is hereby incorporated by reference in their entirety:

Type Risk Factors Prerenal ECF volume depletion Excessive diuresis, hemorrhage, GI losses, loss of intravascular fluid into the extravascular space (due to ascites, peritonitis, pancreatitis, or burns), loss of skin and mucus membranes, renal salt- and water-wasting states Low cardiac output Cardiomyopathy, MI, cardiac tamponade, pulmonary embolism, pulmonary hypertension, positive-pressure mechanical ventilation Low systemic vascular Septic shock, liver failure, antihypertensive drugs resistance Increased renal vascular NSAIDs, cyclosporines, tacrolimus, hypercalcemia, resistance anaphylaxis, anesthetics, renal artery obstruction, renal vein thrombosis, sepsis, hepatorenal syndrome Decreased efferent ACE inhibitors or angiotensin II receptor blockers arteriolar tone (leading to decreased GFR from reduced glomerular transcapillary pressure, especially in patients with bilateral renal artery stenosis) Intrinsic Renal Acute tubular injury Ischemia (prolonged or severe prerenal state): surgery, hemorrhage, arterial or venous obstruction; Toxins: NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, streptozotocin Acute glomerulonephritis ANCA-associated: Crescentic glomerulonephritis, polyarteritis nodosa, Wegener's granulomatosis; Anti- GBM glomerulonephritis: Goodpasture's syndrome; Immune-complex: Lupus glomerulonephritis, postinfectious glomerulonephritis, cryoglobulinemic glomerulonephritis Acute tubulointerstitial Drug reaction (eg, β-lactams, NSAIDs, sulfonamides, nephritis ciprofloxacin, thiazide diuretics, furosemide, phenytoin, allopurinol, pyelonephritis, papillary necrosis Acute vascular Vasculitis, malignant hypertension, thrombotic nephropathy microangiopathies, scleroderma, atheroembolism Infiltrative diseases Lymphoma, sarcoidosis, leukemia Postrenal Tubular precipitation Uric acid (tumor lysis), sulfonamides, triamterene, acyclovir, indinavir, methotrexate, ethylene glycol ingestion, myeloma protein, myoglobin Ureteral obstruction Intrinsic: Calculi, clots, sloughed renal tissue, fungus ball, edema, malignancy, congenital defects; Extrinsic: Malignancy, retroperitoneal fibrosis, ureteral trauma during surgery or high impact injury Bladder obstruction Mechanical: Benign prostatic hyperplasia, prostate cancer, bladder cancer, urethral strictures, phimosis, paraphimosis, urethral valves, obstructed indwelling urinary catheter; Neurogenic: Anticholinergic drugs, upper or lower motor neuron lesion

In the case of ischemic ARF, the course of the disease may be divided into four phases. During an initiation phase, which lasts hours to days, reduced perfusion of the kidney is evolving into injury. Glomerular ultrafiltration reduces, the flow of filtrate is reduced due to debris within the tubules, and back leakage of filtrate through injured epithelium occurs. Renal injury can be mediated during this phase by reperfusion of the kidney. Initiation is followed by an extension phase which is characterized by continued ischemic injury and inflammation and may involve endothelial damage and vascular congestion. During the maintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs, and glomerular filtration and urine output reaches a minimum. A recovery phase can follow in which the renal epithelium is repaired and GFR gradually recovers. Despite this, the survival rate of subjects with ARF may be as low as about 60%.

Acute kidney injury caused by radiocontrast agents (also called contrast media) and other nephrotoxins such as cyclosporine, antibiotics including aminoglycosides and anticancer drugs such as cisplatin manifests over a period of days to about a week. Contrast induced nephropathy (CIN, which is AKI caused by radiocontrast agents) is thought to be caused by intrarenal vasoconstriction (leading to ischemic injury) and from the generation of reactive oxygen species that are directly toxic to renal tubular epithelial cells. CIN classically presents as an acute (onset within 24-48 h) but reversible (peak 3-5 days, resolution within 1 week) rise in blood urea nitrogen and serum creatinine.

A commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 days or within a period of hospitalization) elevation of serum creatinine. Although the use of serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum creatinine elevation and the time over which it is measured to define AKI varies considerably among publications. Traditionally, relatively large increases in serum creatinine such as 100%, 200%, an increase of at least 100% to a value over 2 mg/dL and other definitions were used to define AKI. However, the recent trend has been towards using smaller serum creatinine rises to define AKI. The relationship between serum creatinine rise, AKI and the associated health risks are reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270, 2005 and Chertow et al, J Am Soc Nephrol 16: 3365-3370, 2005, which, with the references listed therein, are hereby incorporated by reference in their entirety. As described in these publications, acute worsening renal function (AKI) and increased risk of death and other detrimental outcomes are now known to be associated with very small increases in serum creatinine. These increases may be determined as a relative (percent) value or a nominal value. Relative increases in serum creatinine as small as 20% from the pre-injury value have been reported to indicate acutely worsening renal function (AKI) and increased health risk, but the more commonly reported value to define AKI and increased health risk is a relative increase of at least 25%. Nominal increases as small as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported to indicate worsening renal function and increased risk of death. Various time periods for the serum creatinine to rise to these threshold values have been used to define AKI, for example, ranging from 2 days, 3 days, 7 days, or a variable period defined as the time the patient is in the hospital or intensive care unit. These studies indicate there is not a particular threshold serum creatinine rise (or time period for the rise) for worsening renal function or AKI, but rather a continuous increase in risk with increasing magnitude of serum creatinine rise.

One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, hereby incorporated by reference in its entirety) investigated both increases and decreases in serum creatinine. Patients with a mild fall in serum creatinine of −0.1 to −0.3 mg/dL following heart surgery had the lowest mortality rate. Patients with a larger fall in serum creatinine (more than or equal to −0.4 mg/dL) or any increase in serum creatinine had a larger mortality rate. These findings caused the authors to conclude that even very subtle changes in renal function (as detected by small creatinine changes within 48 hours of surgery) seriously effect patient's outcomes. In an effort to reach consensus on a unified classification system for using serum creatinine to define AKI in clinical trials and in clinical practice, Bellomo et al., Crit Care. 8(4):R204-12, 2004, which is hereby incorporated by reference in its entirety, proposes the following classifications for stratifying AKI patients:

“Risk”: serum creatinine increased 1.5 fold from baseline OR urine production of <0.5 ml/kg body weight/hr for 6 hours; “Injury”: serum creatinine increased 2.0 fold from baseline OR urine production <0.5 ml/kg/hr for 12 h; “Failure”: serum creatinine increased 3.0 fold from baseline OR creatinine >355 μmol/l (with a rise of >44) or urine output below 0.3 ml/kg/hr for 24 h or anuria for at least 12 hours; And included two clinical outcomes: “Loss”: persistent need for renal replacement therapy for more than four weeks. “ESRD”: end stage renal disease—the need for dialysis for more than 3 months. These criteria are called the RIFLE criteria, which provide a useful clinical tool to classify renal status. As discussed in Kellum, Crit. Care Med. 36: S141-45, 2008 and Ricci et al., Kidney Int. 73, 538-546, 2008, each hereby incorporated by reference in its entirety, the RIFLE criteria provide a uniform definition of AKI which has been validated in numerous studies.

More recently, Mehta et al., Crit. Care 11:R31 (doi:10.1186.cc5713), 2007, hereby incorporated by reference in its entirety, proposes the following similar classifications for stratifying AKI patients, which have been modified from RIFLE:

“Stage I”: increase in serum creatinine of more than or equal to 0.3 mg/dL (>26.4 μmol/L) or increase to more than or equal to 150% (1.5-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 6 hours; “Stage II”: increase in serum creatinine to more than 200% (>2-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 12 hours; “Stage III”: increase in serum creatinine to more than 300% (>3-fold) from baseline OR serum creatinine ≥354 μmol/L accompanied by an acute increase of at least 44 μmol/L OR urine output less than 0.3 mL/kg per hour for 24 hours or anuria for 12 hours.

The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med. 2006; 7(4):177-197, hereby incorporated by reference in its entirety) uses a serum creatinine rise of 25% to define Contrast induced nephropathy (which is a type of AKI). Although various groups propose slightly different criteria for using serum creatinine to detect AKI, the consensus is that small changes in serum creatinine, such as 0.3 mg/dL or 25%, are sufficient to detect AKI (worsening renal function) and that the magnitude of the serum creatinine change is an indicator of the severity of the AKI and mortality risk.

Although serial measurement of serum creatinine over a period of days is an accepted method of detecting and diagnosing AKI and is considered one of the most important tools to evaluate AKI patients, serum creatinine is generally regarded to have several limitations in the diagnosis, assessment and monitoring of AKI patients. The time period for serum creatinine to rise to values (e.g., a 0.3 mg/dL or 25% rise) considered diagnostic for AKI can be 48 hours or longer depending on the definition used. Since cellular injury in AKI can occur over a period of hours, serum creatinine elevations detected at 48 hours or longer can be a late indicator of injury, and relying on serum creatinine can thus delay diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of the exact kidney status and treatment needs during the most acute phases of AKI when kidney function is changing rapidly. Some patients with AKI will recover fully, some will need dialysis (either short term or long term) and some will have other detrimental outcomes including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration rate, it does not differentiate between the causes of AKI (pre-renal, intrinsic renal, post-renal obstruction, atheroembolic, etc) or the category or location of injury in intrinsic renal disease (for example, tubular, glomerular or interstitial in origin). Urine output is similarly limited, Knowing these things can be of vital importance in managing and treating patients with AKI.

These limitations underscore the need for better methods to detect and assess AKI, particularly in the early and subclinical stages, but also in later stages when recovery and repair of the kidney can occur. Furthermore, there is a need to better identify patients who arc at risk of having an AKI.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to provide methods and compositions for evaluating renal function in a subject. As described herein, measurement of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and Insulin-like growth factor-binding protein 6 (collectively referred to herein as “kidney injury markers, and individually as a “kidney injury marker”) can be used for diagnosis, prognosis, risk stratification, staging, monitoring, categorizing and determination of further diagnosis and treatment regimens in subjects suffering or at risk of suffering from an injury to renal function, reduced renal function, and/or acute renal failure (also called acute kidney injury).

These kidney injury markers may be used, individually or in panels comprising a plurality of kidney injury markers, for risk stratification (that is, to identify subjects at risk for a future injury to renal function, for future progression to reduced renal function, for future progression to ARF, for future improvement in renal function, etc.); for diagnosis of existing disease (that is, to identify subjects who have suffered an injury to renal function, who have progressed to reduced renal function, who have progressed to ARF, etc.); for monitoring for deterioration or improvement of renal function; and for predicting a future medical outcome, such as improved or worsening renal function, a decreased or increased mortality risk, a decreased or increased risk that a subject will require renal replacement therapy (i.e., hemodialysis, peritoneal dialysis, hemofiltration, and/or renal transplantation, a decreased or increased risk that a subject will recover from an injury to renal function, a decreased or increased risk that a subject will recover from ARF, a decreased or increased risk that a subject will progress to end stage renal disease, a decreased or increased risk that a subject will progress to chronic renal failure, a decreased or increased risk that a subject will suffer rejection of a transplanted kidney, etc.

In a first aspect, the present invention relates to methods for evaluating renal status in a subject. These methods comprise performing an assay method that is configured to detect one or more kidney injury markers of the present invention in a body fluid sample obtained from the subject. The assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are then correlated to the renal status of the subject. This correlation to renal status may include correlating the assay result(s) to one or more of risk stratification, diagnosis, prognosis, staging, classifying and monitoring of the subject as described herein. Thus, the present invention utilizes one or more kidney injury markers of the present invention for the evaluation of renal injury.

In certain embodiments, the methods for evaluating renal status described herein are methods for risk stratification of the subject; that is, assigning a likelihood of one or more future changes in renal status to the subject. In these embodiments, the assay result(s) is/are correlated to one or more such future changes. The following are preferred risk stratification embodiments.

In preferred risk stratification embodiments, these methods comprise determining a subject's risk for a future injury to renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to a likelihood of such a future injury to renal function. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In other preferred risk stratification embodiments, these methods comprise determining a subject's risk for future reduced renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to a likelihood of such reduced renal function. For example, the measured concentrations may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of suffering a future reduced renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of future reduced renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In still other preferred risk stratification embodiments, these methods comprise determining a subject's likelihood for a future improvement in renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to a likelihood of such a future improvement in renal function. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold. For a “negative going” kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold.

In yet other preferred risk stratification embodiments, these methods comprise determining a subject's risk for progression to ARF, and the result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to a likelihood of such progression to ARF. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

And in other preferred risk stratification embodiments, these methods comprise determining a subject's outcome risk, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to a likelihood of the occurrence of a clinical outcome related to a renal injury suffered by the subject. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In such risk stratification embodiments, preferably the likelihood or risk assigned is that an event of interest is more or less likely to occur within 180 days of the time at which the body fluid sample is obtained from the subject. In particularly preferred embodiments, the likelihood or risk assigned relates to an event of interest occurring within a shorter time period such as 18 months, 120 days, 90 days, 60 days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, or less. A risk at 0 hours of the time at which the body fluid sample is obtained from the subject is equivalent to diagnosis of a current condition.

In preferred risk stratification embodiments, the subject is selected for risk stratification based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF. For example, a subject undergoing or having undergone major vascular surgery, coronary artery bypass, or other cardiac surgery; a subject having pre-existing congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, glomerular filtration below the normal range, cirrhosis, serum creatinine above the normal range, or sepsis; or a subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin are all preferred subjects for monitoring risks according to the methods described herein. This list is not meant to be limiting. By “pre-existence” in this context is meant that the risk factor exists at the time the body fluid sample is obtained from the subject. In particularly preferred embodiments, a subject is chosen for risk stratification based on an existing diagnosis of injury to renal function, reduced renal function, or ARF.

In other embodiments, the methods for evaluating renal status described herein are methods for diagnosing a renal injury in the subject; that is, assessing whether or not a subject has suffered from an injury to renal function, reduced renal function, or ARF. In these embodiments, the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status. The following are preferred diagnostic embodiments.

In preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of an injury to renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the occurrence or nonoccurrence of such an injury. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury to renal function may he assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In other preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of reduced renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the occurrence or nonoccurrence of an injury causing reduced renal function. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury causing reduced renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In yet other preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or nonoccurrence of ARF, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the occurrence or nonoccurrence of an injury causing ARF. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of ARF is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprise diagnosing a subject as being in need of renal replacement therapy, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to a need for renal replacement therapy. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprise diagnosing a subject as being in need of renal transplantation, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to a need for renal transplantation. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In still other embodiments, the methods for evaluating renal status described herein are methods for monitoring a renal injury in the subject; that is, assessing whether or not renal function is improving or worsening in a subject who has suffered from an injury to renal function, reduced renal function, or ARF. In these embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/arc correlated to the occurrence or nonoccurrence of a change in renal status. The following arc preferred monitoring embodiments.

In preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from an injury to renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from reduced renal function, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from acute renal failure, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In other additional preferred monitoring embodiments, these methods comprise monitoring renal status in a subject at risk of an injury to renal function due to the pre-existence of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject having, or at risk of, an injury to renal function for future persistence of acute kidney injury. “Future persistence” as used herein refers to an existing acute renal injury that will continue for a period selected from the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, and 12 hours. In certain embodiments the subject has an acute kidney injury at the time the sample is obtained. This is not meant to imply that the subject must have an acute kidney injury at the time the sample is obtained, but rather that the subject, upon onset of an acute kidney injury, suffers from an acute kidney injury that will persist. In various embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/are correlated to the future persistence of the acute kidney injury in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a future persistence of acute kidney injury may be assigned to the subject; alternatively, when the measured concentration is below the threshold, a future improvement of renal function may be assigned to the subject. For a negative going marker, when the measured concentration is below the threshold, a future persistence of acute kidney injury may be assigned to the subject; alternatively, when the measured concentration is above the threshold, a future improvement of renal function may be assigned to the subject.

In still other embodiments, the methods for evaluating renal status described herein are methods for classifying a renal injury in the subject; that is, determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage. In these embodiments, the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/arc correlated to a particular class and/or subclass. The following arc preferred classification embodiments.

In preferred classification embodiments, these methods comprise determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage, and the assay result(s), for example a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and/or Insulin-like growth factor-binding protein 6, is/arc correlated to the injury classification for the subject. For example, the measured concentration may be compared to a threshold value, and when the measured concentration is above the threshold, a particular classification is assigned; alternatively, when the measured concentration is below the threshold, a different classification may be assigned to the subject.

A variety of methods may be used by the skilled artisan to arrive at a desired threshold value for use in these methods. For example, the threshold value may be determined from a population of normal subjects by selecting a concentration representing the 75^(th), 85^(th), 90^(th), 95^(th), or 99^(th) percentile of a kidney injury marker measured in such normal subjects. Alternatively, the threshold value may be determined from a “diseased” population of subjects, e.g., those suffering from an injury or having a predisposition for an injury (e.g., progression to ARF or some other clinical outcome such as death, dialysis, renal transplantation, etc.), by selecting a concentration representing the 75^(th), 85^(th), 90^(th), 95^(th), or 99^(th) percentile of a kidney injury marker measured in such subjects. In another alternative, the threshold value may be determined from a prior measurement of a kidney injury marker in the same subject; that is, a temporal change in the level of a kidney injury marker in the subject may be used to assign risk to the subject.

The foregoing discussion is not meant to imply, however, that the kidney injury markers of the present invention must be compared to corresponding individual thresholds. Methods for combining assay results can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, calculating ratios of markers, etc. This list is not meant to be limiting. In these methods, a composite result which is determined by combining individual markers may be treated as if it is itself a marker; that is, a threshold may be determined for the composite result as described herein for individual markers, and the composite result for an individual patient compared to this threshold.

The ability of a particular test to distinguish two populations can be established using ROC analysis. For example, ROC curves established from a “first” subpopulation which is predisposed to one or more future changes in renal status, and a “second” subpopulation which is not so predisposed can be used to calculate a ROC curve, and the area under the curve provides a measure of the quality of the test. Preferably, the tests described herein provide a ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95.

In certain aspects, the measured concentration of one or more kidney injury markers, or a composite of such markers, may be treated as continuous variables. For example, any particular concentration can be converted into a corresponding probability of a future reduction in renal function for the subject, the occurrence of an injury, a classification, etc. In yet another alternative, a threshold that can provide an acceptable level of specificity and sensitivity in separating a population of subjects into “bins” such as a “first” subpopulation (e.g., which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc.) and a “second” subpopulation which is not so predisposed. A threshold value is selected to separate this first and second population by one or more of the following measures of test accuracy:

an odds ratio greater than 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a specificity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95; a sensitivity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably at least about 0.95, with a corresponding specificity greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95; at least about 75% sensitivity, combined with at least about 75% specificity; a positive likelihood ratio (calculated as sensitivity/(1-specificity)) of greater than 1, at least about 2, more preferably at least about 3, still more preferably at least about 5, and most preferably at least about 10; or a negative likelihood ratio (calculated as (1-sensitivity)/specificity) of less than 1, less than or equal to about 0.5, more preferably less than or equal to about 0.3, and most preferably less than or equal to about 0.1. The term “about” in the context of any of the above measurements refers to +/−5% of a given measurement.

Multiple thresholds may also be used to assess renal status in a subject. For example, a “first” subpopulation which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc., and a “second” subpopulation which is not so predisposed can be combined into a single group. This group is then subdivided into three or more equal parts (known as tertiles, quartiles, quintiles, etc., depending on the number of subdivisions). An odds ratio is assigned to subjects based on which subdivision they fall into. If one considers a tertile, the lowest or highest tertile can be used as a reference for comparison of the other subdivisions. This reference subdivision is assigned an odds ratio of 1. The second tertile is assigned an odds ratio that is relative to that first tertile. That is, someone in the second tertile might be 3 times more likely to suffer one or more future changes in renal status in comparison to someone in the first tertile. The third tertile is also assigned an odds ratio that is relative to that first tertile.

In certain embodiments, the assay method is an immunoassay. Antibodies for use in such assays will specifically bind a full length kidney injury marker of interest, and may also bind one or more polypeptides that are “related” thereto, as that term is defined hereinafter. Numerous immunoassay formats are known to those of skill in the art. Preferred body fluid samples are selected from the group consisting of urine, blood, serum, saliva, tears, and plasma.

The foregoing method steps should not be interpreted to mean that the kidney injury marker assay result(s) is/are used in isolation in the methods described herein. Rather, additional variables or other clinical indicia may be included in the methods described herein. For example, a risk stratification, diagnostic, classification, monitoring, etc. method may combine the assay result(s) with one or more variables measured for the subject selected from the group consisting of demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, a renal failure index calculated as urine sodium/(urine creatinine/plasma creatinine), a serum or plasma neutrophil gelatinase (NGAL) concentration, a urine NGAL concentration, a serum or plasma cystatin C concentration, a serum or plasma cardiac troponin concentration, a serum or plasma BNP concentration, a serum or plasma NTproBNP concentration, and a serum or plasma proBNP concentration. Other measures of renal function which may be combined with one or more kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill, N.Y., pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, N.Y., pages 785-815, each of which are hereby incorporated by reference in their entirety.

When more than one marker is measured, the individual markers may be measured in samples obtained at the same time, or may be determined from samples obtained at different (e.g., an earlier or later) times. The individual markers may also be measured on the same or different body fluid samples. For example, one kidney injury marker may be measured in a serum or plasma sample and another kidney injury marker may be measured in a urine sample. In addition, assignment of a likelihood may combine an individual kidney injury marker assay result with temporal changes in one or more additional variables.

In various related aspects, the present invention also relates to devices and kits for performing the methods described herein. Suitable kits comprise reagents sufficient for performing an assay for at least one of the described kidney injury markers, together with instructions for performing the described threshold comparisons.

In certain embodiments, reagents for performing such assays are provided in an assay device, and such assay devices may be included in such a kit. Preferred reagents can comprise one or more solid phase antibodies, the solid phase antibody comprising antibody that detects the intended biomarker target(s) bound to a solid support. In the case of sandwich immunoassays, such reagents can also include one or more detectably labeled antibodies, the detectably labeled antibody comprising antibody that detects the intended biomarker target(s) bound to a detectable label. Additional optional elements that may be provided as part of an assay device arc described hereinafter.

Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, ecl (electrochemical luminescence) labels, metal chelates, colloidal metal particles, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or through the use of a specific binding molecule which itself may be detectable (e.g., a labeled antibody that binds to the second antibody, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Generation of a signal from the signal development element can be performed using various optical, acoustical, and electrochemical methods well known in the art. Examples of detection modes include fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductance, impedance, interferometry, ellipsometry, etc. In certain of these methods, the solid phase antibody is coupled to a transducer (e.g., a diffraction grating, electrochemical sensor, etc) for generation of a signal, while in others, a signal is generated by a transducer that is spatially separate from the solid phase antibody (e.g., a fluorometer that employs an excitation light source and an optical detector). This list is not meant to be limiting. Antibody-based biosensors may also be employed to determine the presence or amount of analytes that optionally eliminate the need for a labeled molecule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in subjects suffering or at risk of suffering from injury to renal function, reduced renal function and/or acute renal failure through measurement of one or more kidney injury markers. In various embodiments, a measured concentration of one or more markers selected from the group consisting of Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and Insulin-like growth factor-binding protein 6 or one or more markers related thereto, and optionally one or more additional kidney injury markers known in the art, are correlated to the renal status of the subject.

For purposes of this document, the following definitions apply:

As used herein, an “injury to renal function” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable reduction in a measure of renal function. Such an injury may be identified, for example, by a decrease in glomerular filtration rate or estimated GFR, a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy, etc. “Improvement in Renal Function” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable increase in a measure of renal function. Preferred methods for measuring and/or estimating GFR arc described hereinafter. As used herein, “reduced renal function” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.1 mg/dL (≥8.8 μmol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour).

As used herein, “acute renal failure” or “ARF” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.3 mg/dl (≥26.4 μmol/l), a percentage increase in serum creatinine of greater than or equal to 50% (1.5-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour for at least 6 hours). This term is synonymous with “acute kidney injury” or “AKI.”

In this regard, the skilled artisan will understand that the signals obtained from an immunoassay arc a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length biomarker and the assay result he expressed as a concentration of a biomarker of interest, the signal from the assay is actually a result of all such “immunoreactive” polypeptides present in the sample. Expression of biomarkers may also be determined by means other than immunoassays, including protein measurements (such as dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quatitation). This list is not meant to be limiting.

As used herein, the term “Insulin-like growth factor-binding protein 3” refers to one or more polypeptides present in a biological sample that are derived from a Insulin-like growth factor-binding protein 3 precursor (human precursor Swiss-Prot entry P17936 (SEQ ID NO: 1)):

        10         20         30         40 MQRARPTLWA AALTLLVLLR GPPVARAGAS SAGLGPVVRC         50         60         70         80 EPCDARALAQ CAPPPAVCAE LVREPGCGCC LTCALSEGQP         90        100        110        120 CGIYTERCGS GLRCQPSPDE ARPLQALLDG RGLCVNASAV        130        140        150        160 SRLRAYLLPA PPAPGNASES EEDRSAGSVE SPSVSSTHRV        170        180        190        200 SDPKFHPLHS KIIIIKKGHA KDSQRYKVDY ESQSTDTQNF        210        220        230        240 SSESKRETEY GPCRREMEDT LNHLKFLNVL SPRGVHIPNC        250        260        270        280 DKKGFYKKKQ CRPSKGRKRG FCWCVDKYGQ PLPGYTTKGK        290 EDVHCYSMQS K

The following domains have been identified in Insulin-like growth factor-binding protein 3:

Residues Length Domain ID 1-27  27 signal sequence 28-291 264 Insulin-like growth factor-binding protein 3 135-135 G→GEPPAPG (SEQ ID NO: 2) in isoform 2

As used herein, the term “Insulin-like growth factor-binding protein 2” refers to one or more polypeptides present in a biological sample that are derived from a Insulin-like growth factor-binding protein 2 precursor (human precursor Swiss-Prot entry P18065) (SEQ ID NO: 3)):

        10         20         30         40 MLPRVGCPAL PLPPPPLLPL LLLLLGASGG GGGARAEVLF         50         60         70         80 RCPPCTPERL AACGPPPVAP PAAVAAVAGG ARMPCAELVR         90        100        110        120 EPGCGCCSVC ARLEGEACGV YTPRCGQGLR CYPHPGSELP        130        140        150        160 LQALVMGEGT CEKRRDAEYG ASPEQVADNG DDHSEGGLVE        170        180        190        200 NHVDSTMNML GGGGSAGRKP LKSGMKELAV FREKVTEQHR        210        220        230        240 QMGKGGKHHL GLEEPKKLRP PPARTPCQQE LDQVLERIST        250        260        270        280 MRLPDERGPL EHLYSLHIPN CDKHGLYNLK QCKMSLNGQR        290        300        310        320 GECWCVNPNT GKLIQGAPTI RGDPECHLFY NEQQEARGVH TQRMQ

The following domains have been identified in Insulin-like growth factor-binding protein 2:

Residues Length Domain ID 1-35  35 signal sequence 36-325 290 Insulin-like growth factor-binding protein 2

As used herein, the term “Insulin-like growth factor-binding protein 4” refers to one or more polypeptides present in a biological sample that are derived from a Insulin-like growth factor-binding protein 4 precursor (human precursor Swiss-Prot entry P22692(SEQ ID NO: 4)):

        10         20         30         40 MLPLCLVAAL LLAAGPGPSL GDEAIHCPPC SEEKLARCRP         50         60         70         80 PVGCEELVRE PGCGCCATCA LGLGMPCGVY TPRCGSGLRC         90        100        110        120 YPPRGVEKPL HTLMHGQGVC MELAEIEAIQ ESLQPSDKDE        130        140        150        160 GDHPNNSFSP CSAHDRRCLQ KHFAKIRDRS TSGGKMKVNG        170        180        190        200 APREDARPVP QGSCQSELHR ALERLAASQS RTHEDLYIIP        210        220        230        240 IPNCDRNGNF HPKQCHPALD GQRGKCWCVD RKTGVKLPGG        250 LEPKGELDCH QLADSFRE

The following domains have been identified in Insulin-like growth factor-binding protein 4:

Residues Length Domain ID 1-21  21 signal sequence 22-258 237 Insulin-like growth factor-binding protein 4  1-100 missing in isoform 2

As used herein, the term “Insulin-like growth factor-binding protein 6” refers to one or more polypeptides present in a biological sample that are derived from a Insulin-like growth factor-binding protein 6 precursor (human precursor Swiss-Prot entry P24592 (SEQ ID NO: 5)):

        10         20         30         40 MTPHRLLPPL LLLLALLLAA SPGGALARCP GCGQGVQAGC         50         60         70         80 PGGCVEEEDG GSPAEGCAEA EGCLRREGQE CGVYTPNCAP         90        100        110        120 GLQCHPPKDD EAPLRALLLG RGRCLPARAP AVAEENPKES        130        140        150        160 KPQAGTARPQ DVNRRDQQRN PGTSTTPSQP NSAGVQDTEM        170        180        190        200 GPCRRHLDSV LQQLQTEVYR GAQTLYVPNC DHRGFYRKRQ        210        220        230        240 CRSSQGQRRG PCWCVDRMGK SLPGSPDGNG SSSCPTGSSG

The following domains have been identified in Insulin-like growth factor-binding protein 6:

Residues Length Domain ID 1-27  27 signal sequence 28-240 213 Insulin-like growth factor-binding protein 6

As used herein, the term “relating a signal to the presence or amount” of an analyte reflects this understanding. Assay signals are typically related to the presence or amount of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest. As the term is used herein, an assay is “configured to detect” an analyte if an assay can generate a detectable signal indicative of the presence or amount of a physiologically relevant concentration of the analyte. Because an antibody epitope is on the order of 8 amino acids, an immunoassay configured to detect a marker of interest will also detect polypeptides related to the marker sequence, so long as those polypeptides contain the epitope(s) necessary to bind to the antibody or antibodies used in the assay.

The term “related marker” as used herein with regard to a biomarker such as one of the kidney injury markers described herein refers to one or more fragments, variants, etc., of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent biomarkers. The term also refers to one or more polypeptides present in a biological sample that are derived from the biomarker precursor complexed to additional species, such as binding proteins, receptors, heparin, lipids, sugars, etc.

The term “positive going” marker as that term is used herein refer to a marker that is determined to be elevated in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition. The term “negative going” marker as that term is used herein refer to a marker that is determined to be reduced in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.

The term “subject” as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Further, while a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects are humans, and most preferably “patients,” which as used herein refers to living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.

Preferably, an analyte is measured in a sample. Such a sample may be obtained from a subject, or may be obtained from biological materials intended to be provided to the subject. For example, a sample may be obtained from a kidney being evaluated for possible transplantation into a subject, and an analyte measurement used to evaluate the kidney for preexisting damage. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which the skilled artisan can estimate and/or determine the probability (“a likelihood”) of whether or not a patient is suffering from a given disease or condition. In the case of the present invention, “diagnosis” includes using the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the subject from which a sample was obtained and assayed. That such a diagnosis is “determined” is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. The skilled clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the occurrence of disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) that a given course or outcome will occur. A level or a change in level of a prognostic indicator, which in turn is associated with an increased probability of morbidity (e.g., worsening renal function, future ARF, or death) is referred to as being “indicative of an increased likelihood” of an adverse outcome in a patient.

Marker Assays

In general, immunoassays involve contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein “blotting” methods. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among the immunoassay analyzers that are capable of performing immunoassays. But any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays. Solid phases that may be used to immobilize specific binding members include include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.

Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Tmidoesters are also very useful for protein-protein cross-links. A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysis of the described kidney injury markers. The kit comprises reagents for the analysis of at least one test sample which comprise at least one antibody that a kidney injury marker. The kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein. Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labeled species for performing a competitive assay, for the analyte. Preferably, an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies that bind a kidney injury marker. Most preferably each of the antibodies are monoclonal antibodies. The instructions for use of the kit and performing the correlations can be in the form of labeling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W. E. Paul, cd., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., “antigen binding sites,” (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.”

Antibodies used in the immunoassays described herein preferably specifically bind to a kidney injury marker of the present invention. The term “specifically binds” is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). Preferably the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, Preferred antibodies bind with affinities of at least about 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹ M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹² M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is the dissociation rate constant, K_(on), is the association rate constant and K_(d) is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r/c=K(n−r): where r=moles of bound ligand/mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K=equilibrium association constant; and n=number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

The term “epitope” refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.

The antibodies that arc generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.

The antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.

Assay Correlations

The term “correlating” as used herein in reference to the use of biomarkers refers to comparing the presence or amount of the biomarker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition. Often, this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold selected to be indicative of the occurrence or nonoccurrence of a disease or the likelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. For example, when considering administering a specific therapy which is highly efficacious and has a low level of risk, few tests are needed because clinicians can accept substantial diagnostic uncertainty. On the other hand, in situations where treatment options are less effective and more risky, clinicians often need a higher degree of diagnostic certainty. Thus, cost/benefit analysis is involved in selecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5^(th) percentile of the concentration seen in a normal population. Another method may be to look at serial samples from the same patient, where a prior “baseline” result is used to monitor for temporal changes in a biomarker level.

Population studies may also be used to select a decision threshold. Reciever Operating Characteristic (“ROC”) arose from the field of signal detection theory developed during World War II for the analysis of radar images, and ROC analysis is often used to select a threshold able to best distinguish a “diseased” subpopulation from a “nondiseased” subpopulation. A false positive in this case occurs when the person tests positive, but actually does not have the disease. A false negative, on the other hand, occurs when the person tests negative, suggesting they are healthy, when they actually do have the disease. To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1—specificity, the ROC graph is sometimes called the sensitivity vs (1—specificity) plot. A perfect test will have an area under the ROC curve of 1.0; a random test will have an area of 0.5. A threshold is selected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome) and “nondiseased” is meant to refer to a population lacking the characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlating assay results to a patient classification (occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (“AUC”) of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(1−specificity)) of greater than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1−sensitivity)/specificity) of less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1

Additional clinical indicia may be combined with the kidney injury marker assay result(s) of the present invention. These include other biomarkers related to renal status. Examples include the following, which recite the common biomarker name, followed by the Swiss-Prot entry number for that biomarker or its parent: Actin (P68133); Adenosine deaminase binding protein (DPP4, P27487); Alpha-1-acid glycoprotein 1 (P02763); Alpha-1-microglobulin (P02760); Albumin (P02768); Angiotensinogenase (Renin, P00797); Nidogen-1 (P07355); Beta-glucuronidase (P08236); B-2-microglobulin (P61769); Beta-galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide (proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta (S100-beta, P04271); Carbonic anhydrase 9 (Q16790); Casein Kinase 2 (P68400); Clusterin (P10909); Complement C3 (P01024); Cysteine-rich protein (CYR61, 000622); Cytochrome C (P99999); Epidermal growth factor (EGF, P01133); Endothelin-1 (P05305); Exosomal Fetuin-A (P02765); Fatty acid-binding protein, heart (FABP3, P05413); Fatty acid-binding protein, liver (P07148); Ferritin (light chain, P02792; heavy chain P02794); Fructose-1,6-biphosphatase (P09467); GRO-alpha (CXCL1, (P09341); Growth Hormone (P01241); Hepatocyte growth factor (P14210); Insulin-like growth factor I (P05019); Immunoglobulin G; Immunoglobulin Light Chains (Kappa and Lambda); Interferon gamma (P01308); Lysozyme (P61626); Interleukin-1alpha (P01583); Interleukin-2 (P60568); Interleukin-4 (P05112); Interleukin-9 (P15248); Interleukin-12p40 (P29460); Interleukin-13 (P35225); Interleukin-16 (Q14005); L1 cell adhesion molecule (P32004); Lactate dehydrogenase (P00338); Leucine Aminopeptidase (P28838); Meprin A-alpha subunit (Q16819); Meprin A-beta subunit (Q16820); Midkine (P21741); MIP2-alpha (CXCL2, P19875); MMP-2 (P08253); MMP-9 (P14780); Netrin-1 (095631); Neutral endopeptidase (P08473); Osteopontin (014788); Renal papillary antigen 1 (RPA1); Renal papillary antigen 2 (RPA2); Retinol binding protein (P09455); Ribonuclease; S100 calcium-binding protein A6 (P06703); Serum Amyloid P Component (P02743); Sodium/Hydrogen exchanger isoform (NHE3, P48764); Spermidine/spermine N1-acetyltransferase (P21673); TGF-Beta1 (P01137); Transferrin (P02787); Trefoil factor 3 (TFF3, Q07654); Toll-Like protein 4 (000206); Total protein; Tubulointerstitial nephritis antigen (Q9UJW2); Uromodulin (Tamm-Horsfall protein, P07911).

For purposes of risk stratification, Adiponectin (Q15848); Alkaline phosphatase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937); Cystatin C (P01034); 8 subunit of F1FO ATPase (P03928); Gamma-glutamyltransferase (P19440); GSTa (alpha-glutathione-S-transferase, P08263); GSTpi (Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-1 (P08833); IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itm1, P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18 (Q14116); IP-10 (10 kDa interferon-gamma-induced protein, P02778); IRPR (IFRD1, 000458); Isovaleryl-CoA dehydrogenase (IVD, P26440); I-TAC/CXCL11 (014625); Keratin 19 (P08727); Kim-1 (Hepatitis A virus cellular receptor 1, 043656); L-arginine:glycine amidinotransferase (P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-1 (P13500); MIG (Gamma-interferon-induced monokine Q07325); MIP-1a (P10147); MIP-3a (P78556); MIP-1beta (P13236); MIP-1d (Q16663); NAG (N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter (OCT2, 015244); Osteoprotegerin (014788); P8 protein (060356); Plasminogen activator inhibitor 1 (PAI-1, P05121); ProANP(1-98) (P01160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab GDI-beta (P50395); Renal kallikrein (P06870); RT1.B-1 (alpha) chain of the integral membrane protein (Q5Y7A8); soluble tumor necrosis factor receptor superfamily member 1A (sTNFR-I, P19438); soluble tumor necrosis factor receptor superfamily member 1B (sTNFR-II, P20333); Tissue inhibitor of metalloproteinases 3 (TIMP-3, P35625); uPAR (Q03405) may be combined with the kidney injury marker assay result(s) of the present invention.

Other clinical indicia which may be combined with the kidney injury marker assay result(s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urine total protein measurement, a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a renal papillary antigen 1 (RPA1) measurement; a renal papillary antigen 2 (RPA2) measurement; a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal failure index calculated as urine sodium/(urine creatinine/plasma creatinine). Other measures of renal function which may be combined with the kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17^(th) Ed., McGraw Hill, N.Y., pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47^(th) Ed, McGraw Hill, N.Y., pages 785-815, each of which are hereby incorporated by reference in their entirety.

Combining assay results/clinical indicia in this manner can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, etc. This list is not meant to be limiting.

Diagnosis of Acute Renal Failure

As noted above, the terms “acute renal (or kidney) injury” and “acute renal (or kidney) failure” as used herein are defined in part in terms of changes in serum creatinine from a baseline value. Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by assuming the patient initially had a normal GFR. Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min:

${GFR} = \frac{{Urine}\mspace{14mu}{Concentration} \times {Urine}\mspace{14mu}{Flow}}{{Plasma}\mspace{14mu}{Concentration}}$

By normalizing the GFR to the body surface area, a GFR of approximately 75-100 ml/min per 1.73 m² can be assumed. The rate therefore measured is the quantity of the substance in the urine that originated from a calculable volume of blood.

There are several different techniques used to calculate or estimate the glomerular filtration rate (GFR or eGFR). In clinical practice, however, creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the renal tubules in very small amounts such that creatinine clearance overestimates actual GFR by 10-20%. This margin of error is acceptable considering the ease with which creatinine clearance is measured.

Creatinine clearance (CCr) can be calculated if values for creatinine's urine concentration (U_(Cr)), urine flow rate (V), and creatinine's plasma concentration (Per) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (U_(Cr)×V) divided by its plasma concentration. This is commonly represented mathematically as:

$C_{Cr} = \frac{U_{Cr} \times V}{P_{Cr}}$

Commonly a 24 hour urine collection is undertaken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken:

$C_{Cr} = \frac{U_{Cr} \times 24\text{-}{hour}\mspace{14mu}{volume}}{P_{Cr} \times 24 \times 60\mspace{14mu}{mins}}$

To allow comparison of results between people of different sizes, the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr corrected for their actual BSA:

$C_{{Cr}\mspace{14mu}{corrected}} = \frac{C_{Cr} \times 1.73}{BSA}$

The accuracy of a creatinine clearance measurement (even when collection is complete) is limited because as glomerular filtration rate (GFR) falls creatinine secretion is increased, and thus the rise in serum creatinine is less. Thus, creatinine excretion is much greater than the filtered load, resulting in a potentially large overestimation of the GFR (as much as a twofold difference). However, for clinical purposes it is important to determine whether renal function is stable or getting worse or better. This is often determined by monitoring serum creatinine alone. Like creatinine clearance, the serum creatinine will not be an accurate reflection of GFR in the non-steady-state condition of ARF. Nonetheless, the degree to which serum creatinine changes from baseline will reflect the change in GFR. Serum creatinine is readily and easily measured and it is specific for renal function.

For purposes of determining urine output on a Urine output on a mL/kg/hr basis, hourly urine collection and measurement is adequate. In the case where, for example, only a cumulative 24-h output was available and no patient weights are provided, minor modifications of the RIFLE urine output criteria have been described. For example, Bagshaw et al., Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an average patient weight of 70 kg, and patients are assigned a RIFLE classification based on the following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h (Failure).

Selecting a Treatment Regimen

Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc. The skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, N J, 1999. In addition, since the methods and compositions described herein provide prognostic information, the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Example 1: Contrast-Induced Nephropathy Sample Collection

The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after receiving intravascular contrast media. Approximately 250 adults undergoing radiographic/angiographic procedures involving intravascular administration of iodinated contrast media are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

males and females 18 years of age or older; undergoing a radiographic/angiographic procedure (such as a CT scan or coronary intervention) involving the intravascular administration of contrast media; expected to be hospitalized for at least 48 hours after contrast administration. able and willing to provide written informed consent for study participation and to comply with all study procedures.

Exclusion Criteria

renal transplant recipients; acutely worsening renal function prior to the contrast procedure; already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; expected to undergo a major surgical procedure (such as involving cardiopulmonary bypass) or an additional imaging procedure with contrast media with significant risk for further renal insult within the 48 hrs following contrast administration; participation in an interventional clinical study with an experimental therapy within the previous 30 days; known infection with human immunodeficiency virus (HIV) or a hepatitis virus.

Immediately prior to the first contrast administration (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL) and a urine sample (10 mL) are collected from each patient. Blood and urine samples are then collected at 4 (±0.5), 8 (±1), 24 (±2) 48 (±2), and 72 (±2) hrs following the last administration of contrast media during the index contrast procedure. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc.

Serum creatinine is assessed at the site immediately prior to the first contrast administration (after any pre-procedure hydration) and at 4 (±0.5), 8 (±1), 24 (±2) and 48 (±2)), and 72 (±2) hours following the last administration of contrast (ideally at the same time as the study samples are obtained). In addition, each patient's status is evaluated through day 30 with regard to additional serum and urine creatinine measurements, a need for dialysis, hospitalization status, and adverse clinical outcomes (including mortality).

Prior to contrast administration, each patient is assigned a risk based on the following assessment: systolic blood pressure <80 mm Hg=5 points; intra-arterial balloon pump=5 points; congestive heart failure (Class III-IV or history of pulmonary edema)=5 points; age >75 yrs=4 points; hematocrit level <39% for men, <35% for women=3 points; diabetes=3 points; contrast media volume=1 point for each 100 mL; serum creatinine level >1.5 g/dL=4 points OR estimated GFR 40-60 mL/min/1.73 m²=2 points, 20-40 mL/min/1.73 m²=4 points, <20 mL/min/1.73 m²=6 points. The risks assigned are as follows: risk for CIN and dialysis: 5 or less total points=risk of CIN −7.5%, risk of dialysis −0.04%; 6-10 total points=risk of CIN −14%, risk of dialysis −0.12%; 11-16 total points=risk of CIN −26.1%, risk of dialysis −1.09%; >16 total points=risk of CIN −57.3%, risk of dialysis −12.8%.

Example 2: Cardiac Surgery Sample Collection

The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after undergoing cardiovascular surgery, a procedure known to be potentially damaging to kidney function. Approximately 900 adults undergoing such surgery are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

males and females 18 years of age or older; undergoing cardiovascular surgery; Toronto/Ottawa Predictive Risk Index for Renal Replacement risk score of at least 2 (Wijeysundera et al., JAMA 297: 1801-9, 2007); and able and willing to provide written informed consent for study participation and to comply with all study procedures.

Exclusion Criteria

known pregnancy; previous renal transplantation; acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria); already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; currently enrolled in another clinical study or expected to be enrolled in another clinical study within 7 days of cardiac surgery that involves drug infusion or a therapeutic intervention for AKI; known infection with human immunodeficiency virus (HIV) or a hepatitis virus.

Within 3 hours prior to the first incision (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3 mL), and a urine sample (35 mL) are collected from each patient. Blood and urine samples are then collected at 3 (±0.5), 6 (±0.5), 12 (±1), 24 (±2) and 48 (±2) hrs following the procedure and then daily on days 3 through 7 if the subject remains in the hospital. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are frozen and shipped to Astute Medical, Inc., San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc.

Example 3: Acutely Ill Subject Sample Collection

The objective of this study is to collect samples from acutely ill patients. Approximately 900 adults expected to be in the ICU for at least 48 hours will be enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

males and females 18 years of age or older; Study population 1: approximately 300 patients that have at least one of: shock (SBP<90 mmHg and/or need for vasopressor support to maintain MAP>60 mmHg and/or documented drop in SBP of at least 40 mmHg); and sepsis; Study population 2: approximately 300 patients that have at least one of: IV antibiotics ordered in computerized physician order entry (CPOE) within 24 hours of enrollment; contrast media exposure within 24 hours of enrollment; increased Intra-Abdominal Pressure with acute decompensated heart failure; and severe trauma as the primary reason for ICU admission and likely to be hospitalized in the ICU for 48 hours after enrollment; Study population 3: approximately 300 patients expected to be hospitalized through acute care setting (ICU or ED) with a known risk factor for acute renal injury (e.g. sepsis, hypotension/shock (Shock=systolic BP<90 mmHg and/or the need for vasopressor support to maintain a MAP>60 mmHg and/or a documented drop in SBP>40 mmHg), major trauma, hemorrhage, or major surgery); and/or expected to be hospitalized to the ICU for at least 24 hours after enrollment.

Exclusion Criteria

known pregnancy; institutionalized individuals; previous renal transplantation; known acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria); received dialysis (either acute or chronic) within 5 days prior to enrollment or in imminent need of dialysis at the time of enrollment; known infection with human immunodeficiency virus (HIV) or a hepatitis virus; meets only the SBP<90 mmHg inclusion criterion set forth above, and does not have shock in the attending physician's or principal investigator's opinion.

After providing informed consent, an EDTA anti-coagulated blood sample (10 mL) and a urine sample (25-30 mL) are collected from each patient. Blood and urine samples are then collected at 4 (±0.5) and 8 (±1) hours after contrast administration (if applicable); at 12 (±1), 24 (±2), and 48 (±2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc.

Example 4. Immunoassay Format

Analytes are measured using standard sandwich enzyme immunoassay techniques. A first antibody which binds the analyte is immobilized in wells of a 96 well polystyrene microplate. Analyte standards and test samples are pipetted into the appropriate wells and any analyte present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase-conjugated second antibody which binds the analyte is added to the wells, thereby forming sandwich complexes with the analyte (if present) and the first antibody. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution comprising tetramethylbenzidine and hydrogen peroxide is added to the wells. Color develops in proportion to the amount of analyte present in the sample. The color development is stopped and the intensity of the color is measured at 540 nm or 570 nm. An analyte concentration is assigned to the test sample by comparison to a standard curve determined from the analyte standards. Units for Insulin-like growth factor-binding protein 2, Insulin-like growth factor-binding protein 3, Insulin-like growth factor-binding protein 4, and Insulin-like growth factor-binding protein 6 reported herein are ng/mL.

Example 5. Apparently Healthy Donor and Chronic Disease Patient Samples

Human urine samples from donors with no known chronic or acute disease (“Apparently Healthy Donors”) were purchased from two vendors (Golden West Biologicals, Inc., 27625 Commerce Center Dr., Temecula, Calif. 92590 and Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, Va. 23454). The urine samples were shipped and stored frozen at less than −20° C. The vendors supplied demographic information for the individual donors including gender, race (Black/White), smoking status and age.

Human urine samples from donors with various chronic diseases (“Chronic Disease Patients”) including congestive heart failure, coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, diabetes mellitus and hypertension were purchased from Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, Va. 23454. The urine samples were shipped and stored frozen at less than −20 degrees centigrade. The vendor provided a case report form for each individual donor with age, gender, race (Black/White), smoking status and alcohol use, height, weight, chronic disease(s) diagnosis, current medications and previous surgeries.

Example 6. Use of CD97 Antigen for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and a urine samples (25-30 mL) are collected from each patient at enrollment, 4 (±0.5) and 8 (±1) hours after contrast administration (if applicable); at 12 (±1), 24 (±2), and 48 (±2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. CD97 antigen is measured in the earliest samples collected while the patients were in RIFLE I or F by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) within 9 days of enrollment, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

Example 6. Use of Insulin-Like Growth Factor-Binding Protein 2 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 2 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 6.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 1.23 5.74 0.497 5.72 0.497 5.72 Average 4.29 17.7 4.50 17.5 4.50 17.5 Stdev 9.77 26.8 10.3 26.7 10.3 26.7 p (t-test) 0.12 0.15 0.15 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 31.9 112 31.9 112 31.9 112 n (Patient) 10 139 9 140 9 140 sCr only Median 2.09 6.60 2.08 6.52 2.08 6.52 Average 5.77 18.8 5.74 18.7 5.74 18.7 Stdev 11.2 27.6 11.4 27.5 11.4 27.5 p (t-test) 0.028 0.032 0.032 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 46.5 112 46.5 112 46.5 112 n (Patient) 23 126 22 127 22 127 UO only Median 3.17 6.67 2.91 6.67 2.85 6.73 Average 13.4 20.4 12.9 20.2 11.3 21.1 Stdev 22.3 29.5 20.7 29.8 18.2 30.4 p (t-test) 0.11 0.091 0.024 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 102 112 91.0 112 90.4 112 n (Patient) 75 73 67 81 64 84 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.78 0.71 0.61 0.79 0.72 0.62 0.79 0.72 0.63 SE 0.061 0.052 0.046 0.061 0.052 0.046 0.061 0.052 0.045 p Value 4.0E−6 7.8E−5 0.016 2.5E−6 3.2E−5 0.0097 2.5E−6 3.2E−5 0.0033 nCohort Recovered 10 23 75 9 22 67 9 22 64 nCohort Non-recovered 139 126 73 140 127 81 140 127 84 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 77% 77% 81% 77% 77% 81% 77% 77% 81% Specificity 50% 35% 31% 56% 36% 33% 56% 36% 33% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 53% 56% 58% 53% 57% 58% 53% 57% 58% Specificity 90% 83% 57% 89% 86% 60% 89% 86% 61% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 27% 29% 32% 26% 28% 30% 26% 28% 31% Specificity 90% 91% 81% 89% 91% 81% 89% 91% 83% OR Quartile 2 3.34 1.78 1.86 4.22 1.93 2.15 4.22 1.93 2.08 p Value 0.069 0.23 0.11 0.040 0.18 0.048 0.040 0.18 0.058 Lower limit of 95% CI 0.910 0.688 0.870 1.07 0.738 1.01 1.07 0.738 0.976 Upper limit of 95% CI 12.3 4.63 3.99 16.6 5.06 4.59 16.6 5.06 4.41 OR Quartile 3 10.2 6.13 1.82 8.97 8.29 2.05 8.97 8.29 2.18 p Value 0.029 0.0017 0.072 0.041 0.0011 0.033 0.041 0.0011 0.021 Lower limit of 95% CI 1.26 1.97 0.949 1.09 2.33 1.06 1.09 2.33 1.12 Upper limit of 95% CI 83.1 19.1 3.49 73.6 29.4 3.95 73.6 29.4 4.24 OR Quartile 4 3.26 4.20 2.00 2.87 3.96 1.75 2.87 3.96 2.16 p Value 0.27 0.061 0.074 0.33 0.073 0.16 0.33 0.073 0.058 Lower limit of 95% CI 0.400 0.936 0.935 0.348 0.879 0.809 0.348 0.879 0.973 Upper limit of 95% CI 26.7 18.8 4.30 23.8 17.8 3.78 23.8 17.8 4.79

TABLE 6.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.21 6.52 2.01 6.39 1.96 6.06 Average 4.52 19.3 4.92 18.8 5.01 18.7 Stdev 6.85 28.0 7.40 27.7 7.57 27.6 p (t-test) 0.0082 0.021 0.026 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 31.9 112 31.9 112 31.9 112 n (Patient) 26 123 22 127 21 128 sCr only Median 2.35 6.86 2.34 6.73 2.22 6.67 Average 5.67 20.5 5.74 20.2 5.83 20.0 Stdev 9.35 28.8 9.62 28.6 9.76 28.6 p (t-test) 0.0034 0.0051 0.0065 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 46.5 112 46.5 112 46.5 112 n (Patient) 35 112 33 114 32 115 UO only Median 3.47 6.78 3.47 6.60 3.17 6.67 Average 12.9 21.5 11.4 21.9 11.0 21.9 Stdev 20.1 31.2 15.6 32.2 15.4 31.8 p (t-test) 0.046 0.015 0.011 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 102 112 73.5 112 73.5 112 n (Patient) 81 63 72 72 69 75 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.69 0.61 0.71 0.69 0.60 0.71 0.69 0.62 SE 0.049 0.047 0.048 0.053 0.048 0.047 0.054 0.048 0.047 p Value 1.1E−5 6.2E−5 0.026 7.8E−5 4.6E−5 0.029 8.3E−5 5.5E−5 0.011 nCohort Recovered 26 35 81 22 33 72 21 32 69 nCohort Non-recovered 123 112 63 127 114 72 128 115 75 Cutoff Quartile 2 1.89 1.91 1.93 1.89 1.91 1.93 1.89 1.91 1.93 Sensitivity 79% 78% 81% 79% 78% 81% 79% 78% 81% Specificity 42% 34% 30% 45% 36% 31% 48% 38% 32% Cutoff Quartile 3 4.13 4.13 4.99 4.13 4.13 4.99 4.13 4.13 4.99 Sensitivity 56% 58% 57% 54% 58% 56% 54% 57% 56% Specificity 77% 74% 56% 73% 76% 56% 71% 75% 57% Cutoff Quartile 4 20.0 20.5 20.2 20.0 20.5 20.2 20.0 20.5 20.2 Sensitivity 30% 31% 32% 29% 31% 31% 29% 30% 31% Specificity 96% 94% 80% 95% 94% 81% 95% 94% 81% OR Quartile 2 2.74 1.82 1.79 3.09 2.03 1.82 3.40 2.16 2.04 p Value 0.027 0.16 0.15 0.019 0.096 0.13 0.012 0.073 0.070 Lower limit of 95% CI 1.12 0.794 0.813 1.20 0.881 0.844 1.31 0.931 0.944 Upper limit of 95% CI 6.66 4.15 3.94 7.91 4.70 3.93 8.85 5.01 4.41 OR Quartile 3 4.26 4.00 1.67 3.17 4.30 1.56 2.92 4.04 1.65 p Value 0.0037 0.0013 0.13 0.024 0.0011 0.18 0.037 0.0019 0.13 Lower limit of 95% CI 1.60 1.71 0.858 1.17 1.78 0.810 1.07 1.67 0.856 Upper limit of 95% CI 11.3 9.31 3.24 8.63 10.3 3.02 8.02 9.76 3.20 OR Quartile 4 10.8 7.50 1.89 8.63 6.87 1.82 8.13 6.56 1.91 p Value 0.022 0.0077 0.10 0.039 0.011 0.13 0.045 0.013 0.10 Lower limit of 95% CI 1.40 1.70 0.882 1.12 1.56 0.844 1.05 1.49 0.875 Upper limit of 95% CI 82.4 33.0 4.05 66.5 30.3 3.93 62.8 29.0 4.15

TABLE 6.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.09 7.04 2.07 7.04 2.07 6.93 Average 3.90 21.2 3.59 20.9 3.72 20.4 Stdev 4.68 29.1 4.29 28.8 4.42 28.6 p (t-test) 4.5E−4 6.8E−4 0.0016 Min 3.16E−5 0.0532 3.16E−5 0.0532 0.242 3.16E−5 Max 17.4 112 17.4 112 17.4 112 n (Patient) 37 109 34 112 31 115 sCr only Median 2.30 8.85 2.09 8.85 2.09 8.85 Average 4.85 22.4 4.61 22.1 4.61 22.1 Stdev 7.76 29.9 7.71 29.6 7.71 29.6 p (t-test) 1.6E−4 2.0E−4 2.0E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 46.5 112 46.5 112 46.5 112 n (Patient) 45 100 43 102 43 102 UO only Median 3.49 6.98 3.34 6.98 3.17 6.67 Average 14.8 21.4 14.7 21.2 15.7 19.6 Stdev 23.3 30.6 23.7 29.9 24.8 28.8 p (t-test) 0.17 0.17 0.42 Min 3.16E−5 0.0645 3.16E−5 0.0645 0.0532 3.16E−5 Max 109 112 109 112 109 112 n (Patient) 70 58 66 62 59 69 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.73 0.60 0.75 0.74 0.61 0.73 0.74 0.59 SE 0.043 0.042 0.050 0.043 0.042 0.050 0.045 0.042 0.050 p Value 5.1E−8 4.0E−8 0.041 9.6E−9 7.9E−9 0.024 2.9E−7 7.9E−9 0.062 nCohort Recovered 37 45 70 34 43 66 31 43 59 nCohort Non-recovered 109 100 58 112 102 62 115 102 69 Cutoff Quartile 2 1.90 1.89 1.89 1.90 1.89 1.89 1.90 1.89 1.89 Sensitivity 79% 81% 83% 79% 81% 84% 78% 81% 83% Specificity 38% 38% 31% 41% 40% 33% 39% 40% 34% Cutoff Quartile 3 4.11 4.13 5.38 4.11 4.13 5.38 4.11 4.13 5.38 Sensitivity 60% 62% 59% 59% 62% 58% 58% 62% 57% Specificity 78% 76% 57% 79% 77% 58% 81% 77% 58% Cutoff Quartile 4 19.7 20.0 22.3 19.7 20.0 22.3 19.7 20.0 22.3 Sensitivity 34% 36% 29% 33% 35% 29% 32% 35% 26% Specificity 100%  98% 79% 100%  98% 79% 100%  98% 76% OR Quartile 2 2.28 2.59 2.20 2.71 2.86 2.60 2.27 2.86 2.44 p Value 0.046 0.017 0.068 0.018 0.0091 0.027 0.058 0.0091 0.034 Lower limit of 95% CI 1.01 1.18 0.942 1.19 1.30 1.11 0.974 1.30 1.07 Upper limit of 95% CI 5.11 5.66 5.14 6.17 6.29 6.07 5.31 6.29 5.55 OR Quartile 3 5.36 5.04 1.89 5.53 5.33 1.88 5.82 5.33 1.77 p Value 1.6E−4 6.1E−5 0.077 2.4E−4 5.4E−5 0.078 3.5E−4 5.4E−5 0.11 Lower limit of 95% CI 2.24 2.29 0.933 2.22 2.37 0.931 2.22 2.37 0.876 Upper limit of 95% CI 12.8 11.1 3.82 13.8 12.0 3.79 15.3 12.0 3.57 OR Quartile 4 38.8 24.8 1.52 34.3 22.9 1.52 30.1 22.9 1.13 p Value 0.011 0.0019 0.31 0.014 0.0024 0.31 0.018 0.0024 0.76 Lower limit of 95% CI 2.32 3.27 0.681 2.04 3.03 0.679 1.79 3.03 0.507 Upper limit of 95% CI 650 187 3.40 575 173 3.40 505 173 2.54

TABLE 6.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.30 7.74 2.20 7.04 2.20 7.04 Average 6.89 21.7 7.15 20.9 7.30 20.6 Stdev 12.2 29.8 12.8 29.3 13.1 29.1 p (t-test) 0.0020 0.0059 0.0091 Min 3.16E−5 0.0532 0.242 3.16E−5 0.242 3.16E−5 Max 56.0 112 56.0 112 56.0 112 n (Patient) 43 99 38 104 36 106 sCr only Median 2.32 9.30 2.32 9.30 2.32 8.42 Average 7.38 22.6 7.38 22.6 7.51 22.2 Stdev 12.7 30.5 12.7 30.5 12.9 30.3 p (t-test) 9.7E−4 9.7E−4 0.0016 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 56.0 112 56.0 112 56.0 112 n (Patient) 50 92 50 92 48 94 UO only Median 3.21 7.04 3.21 6.93 3.53 6.86 Average 15.8 21.0 17.3 19.2 18.1 18.4 Stdev 26.4 28.9 28.0 27.5 28.7 26.8 p (t-test) 0.31 0.71 0.95 Min 3.16E−5 0.0645 0.0532 3.16E−5 0.0532 3.16E−5 Max 109 112 109 112 109 112 n (Patient) 62 57 54 65 50 70 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.70 0.63 0.70 0.70 0.61 0.69 0.69 0.57 SE 0.044 0.044 0.051 0.046 0.044 0.052 0.047 0.044 0.052 p Value 2.4E−6 3.8E−6 0.011 1.8E−5 3.8E−6 0.041 5.2E−5 1.2E−5 0.15 nCohort Recovered 43 50 62 38 50 54 36 48 50 nCohort Non-recovered 99 92 57 104 92 65 106 94 70 Cutoff Quartile 2 1.90 1.90 1.88 1.90 1.90 1.88 1.90 1.90 1.89 Sensitivity 81% 80% 86% 80% 80% 85% 79% 80% 81% Specificity 40% 36% 35% 39% 36% 37% 39% 35% 34% Cutoff Quartile 3 4.99 4.99 5.74 4.99 4.99 5.74 4.99 4.99 6.06 Sensitivity 59% 61% 60% 58% 61% 57% 58% 61% 54% Specificity 70% 70% 58% 71% 70% 57% 72% 71% 56% Cutoff Quartile 4 20.7 20.7 22.6 20.7 20.7 22.6 20.7 20.7 22.3 Sensitivity 33% 35% 30% 32% 35% 26% 31% 34% 26% Specificity 93% 92% 79% 92% 92% 76% 92% 92% 76% OR Quartile 2 2.75 2.31 3.37 2.58 2.31 3.24 2.43 2.16 2.26 p Value 0.012 0.034 0.0089 0.022 0.034 0.0083 0.033 0.051 0.057 Lower limit of 95% CI 1.25 1.07 1.36 1.15 1.07 1.35 1.07 0.996 0.975 Upper limit of 95% CI 6.07 5.01 8.37 5.78 5.01 7.73 5.51 4.71 5.23 OR Quartile 3 3.26 3.63 2.05 3.35 3.63 1.78 3.52 3.74 1.51 p Value 0.0024 5.9E−4 0.055 0.0031 5.9E−4 0.12 0.0028 5.4E−4 0.27 Lower limit of 95% CI 1.52 1.74 0.985 1.50 1.74 0.859 1.54 1.77 0.728 Upper limit of 95% CI 7.01 7.57 4.25 7.46 7.57 3.69 8.04 7.90 3.14 OR Quartile 4 6.67 6.13 1.60 5.42 6.13 1.12 4.97 5.68 1.10 p Value 0.0028 0.0013 0.27 0.0080 0.0013 0.79 0.012 0.0021 0.83 Lower limit of 95% CI 1.92 2.03 0.696 1.55 2.03 0.485 1.42 1.87 0.472 Upper limit of 95% CI 23.2 18.6 3.69 18.9 18.6 2.57 17.4 17.2 2.54

TABLE 6.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 3.09 7.74 3.15 7.39 3.15 7.39 Average 13.8 20.8 13.9 20.5 13.9 20.5 Stdev 23.3 29.3 23.4 29.2 23.4 29.2 p (t-test) 0.10 0.13 0.13 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 86 63 85 64 85 64 sCr only Median 2.65 10.8 2.79 10.2 2.79 10.2 Average 9.32 23.5 9.56 23.0 9.56 23.0 Stdev 15.7 31.5 15.9 31.3 15.9 31.3 p (t-test) 7.8E−4 0.0016 0.0016 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 71 78 69 80 69 80 UO only Median 3.32 7.39 3.47 7.04 3.49 6.91 Average 15.0 20.2 15.3 19.6 15.6 19.0 Stdev 25.1 28.1 25.3 27.8 25.5 27.5 p (t-test) 0.25 0.34 0.45 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 96 52 94 54 92 56 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.62 0.67 0.62 0.62 0.65 0.61 0.62 0.65 0.59 SE 0.047 0.044 0.049 0.047 0.045 0.049 0.047 0.045 0.049 p Value 0.010 9.5E−5 0.016 0.012 7.1E−4 0.026 0.012 7.1E−4 0.058 nCohort Recovered 86 71 96 85 69 94 85 69 92 nCohort Non-recovered 63 78 52 64 80 54 64 80 56 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 81% 81% 83% 81% 79% 83% 81% 79% 82% Specificity 29% 31% 29% 29% 29% 30% 29% 29% 29% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 62% 64% 63% 61% 62% 61% 61% 62% 59% Specificity 58% 65% 57% 58% 64% 56% 58% 64% 55% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 33% 36% 33% 33% 35% 31% 33% 35% 30% Specificity 80% 86% 79% 80% 86% 79% 80% 86% 78% OR Quartile 2 1.74 1.89 1.97 1.81 1.51 2.12 1.81 1.51 1.91 p Value 0.16 0.100 0.12 0.14 0.28 0.080 0.14 0.28 0.12 Lower limit of 95% CI 0.797 0.886 0.847 0.826 0.717 0.915 0.826 0.717 0.843 Upper limit of 95% CI 3.81 4.01 4.57 3.95 3.19 4.92 3.95 3.19 4.33 OR Quartile 3 2.26 3.29 2.33 2.12 2.93 2.03 2.12 2.93 1.78 p Value 0.016 5.2E−4 0.017 0.026 0.0016 0.042 0.026 0.0016 0.091 Lower limit of 95% CI 1.16 1.68 1.16 1.10 1.50 1.03 1.10 1.50 0.911 Upper limit of 95% CI 4.39 6.43 4.67 4.11 5.72 4.02 4.11 5.72 3.50 OR Quartile 4 2.03 3.42 1.85 1.95 3.18 1.70 1.95 3.18 1.57 p Value 0.063 0.0031 0.11 0.078 0.0053 0.17 0.078 0.0053 0.24 Lower limit of 95% CI 0.963 1.51 0.863 0.928 1.41 0.797 0.928 1.41 0.738 Upper limit of 95% CI 4.28 7.70 3.95 4.11 7.16 3.63 4.11 7.16 3.34

Example 7. Use of Insulin-Like Growth Factor-Binding Protein 2 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 2 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 7.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.52 6.86 2.52 6.73 2.44 6.67 Average 6.75 20.1 6.91 19.8 7.01 19.6 Stdev 13.8 28.4 14.1 28.2 14.3 28.2 p (t-test) 0.0068 0.010 0.013 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 37 112 35 114 34 115 sCr only Median 2.79 7.04 2.79 7.04 2.68 7.04 Average 7.47 21.3 7.47 21.3 7.55 21.1 Stdev 13.4 29.5 13.4 29.5 13.5 29.4 p (t-test) 0.0022 0.0022 0.0028 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 49 100 49 100 48 101 UO only Median 3.47 7.04 3.16 6.98 3.15 7.04 Average 14.2 21.4 14.6 20.1 13.9 20.8 Stdev 23.7 29.7 24.5 28.5 23.4 29.3 p (t-test) 0.11 0.21 0.11 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 94 54 88 60 85 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.65 0.62 0.68 0.65 0.62 0.68 0.65 0.63 SE 0.047 0.046 0.049 0.048 0.046 0.047 0.048 0.046 0.047 p Value 1.2E−4 8.0E−4 0.013 2.1E−4 8.0E−4 0.011 1.9E−4 8.0E−4 0.0067 nCohort Recovered 37 49 94 35 49 88 34 48 85 nCohort Non-recovered 112 100 54 114 100 60 115 101 63 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 79% 78% 83% 79% 78% 83% 79% 78% 83% Specificity 38% 31% 30% 37% 31% 31% 38% 31% 31% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 58% 58% 63% 58% 58% 63% 57% 57% 63% Specificity 73% 65% 57% 74% 65% 59% 74% 65% 60% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 32% 35% 35% 32% 35% 32% 31% 35% 32% Specificity 95% 94% 81% 94% 94% 80% 94% 94% 80% OR Quartile 2 2.36 1.56 2.12 2.22 1.56 2.21 2.35 1.63 2.08 p Value 0.038 0.25 0.080 0.057 0.25 0.056 0.043 0.21 0.071 Lower limit of 95% CI 1.05 0.724 0.915 0.976 0.724 0.978 1.03 0.754 0.938 Upper limit of 95% CI 5.28 3.38 4.92 5.03 3.38 5.01 5.36 3.53 4.62 OR Quartile 3 3.73 2.60 2.30 3.97 2.60 2.49 3.74 2.46 2.61 p Value 0.0016 0.0083 0.018 0.0014 0.0083 0.0080 0.0023 0.013 0.0052 Lower limit of 95% CI 1.65 1.28 1.15 1.71 1.28 1.27 1.60 1.21 1.33 Upper limit of 95% CI 8.45 5.29 4.56 9.24 5.29 4.90 8.73 5.01 5.11 OR Quartile 4 8.29 8.26 2.29 7.62 8.26 1.80 7.29 7.95 1.86 p Value 0.0051 8.3E−4 0.032 0.0072 8.3E−4 0.12 0.0086 0.0010 0.11 Lower limit of 95% CI 1.89 2.39 1.07 1.73 2.39 0.850 1.66 2.31 0.878 Upper limit of 95% CI 36.4 28.5 4.90 33.5 28.5 3.82 32.1 27.4 3.94

TABLE 7.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.34 7.81 2.34 7.04 2.34 7.04 Average 6.95 21.9 7.13 21.5 7.13 21.5 Stdev 13.1 29.7 13.3 29.5 13.3 29.5 p (t-test) 8.1E−4 0.0015 0.0015 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 51 98 49 100 49 100 sCr only Median 2.35 10.0 2.34 9.57 2.34 9.57 Average 7.37 22.8 7.44 22.6 7.44 22.6 Stdev 13.6 30.3 13.7 30.2 13.7 30.2 p (t-test) 4.4E−4 5.9E−4 5.9E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 56 91 55 92 55 92 UO only Median 3.49 6.86 3.47 6.93 3.47 6.93 Average 13.8 22.0 13.2 22.2 13.2 22.2 Stdev 22.3 30.8 21.3 31.1 21.3 31.1 p (t-test) 0.072 0.043 0.043 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 94 50 89 55 89 55 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.70 0.61 0.69 0.70 0.61 0.69 0.70 0.61 SE 0.043 0.043 0.050 0.044 0.043 0.049 0.044 0.043 0.049 p Value 4.7E−6 5.3E−6 0.034 1.4E−5 5.3E−6 0.027 1.4E−5 5.3E−6 0.027 nCohort Recovered 51 56 94 49 55 89 49 55 89 nCohort Non-recovered 98 91 50 100 92 55 100 92 55 Cutoff Quartile 2 1.89 1.91 1.93 1.89 1.91 1.93 1.89 1.91 1.93 Sensitivity 81% 79% 82% 80% 79% 80% 80% 79% 80% Specificity 35% 32% 29% 35% 33% 28% 35% 33% 28% Cutoff Quartile 3 4.13 4.13 4.99 4.13 4.13 4.99 4.13 4.13 4.99 Sensitivity 61% 63% 58% 60% 62% 58% 60% 62% 58% Specificity 71% 70% 54% 69% 69% 55% 69% 69% 55% Cutoff Quartile 4 20.0 20.5 20.2 20.0 20.5 20.2 20.0 20.5 20.2 Sensitivity 36% 36% 36% 35% 36% 35% 35% 36% 35% Specificity 94% 93% 81% 94% 93% 81% 94% 93% 81% OR Quartile 2 2.27 1.80 1.84 2.12 1.87 1.56 2.12 1.87 1.56 p Value 0.035 0.13 0.16 0.054 0.11 0.28 0.054 0.11 0.28 Lower limit of 95% CI 1.06 0.844 0.786 0.988 0.877 0.698 0.988 0.877 0.698 Upper limit of 95% CI 4.86 3.82 4.29 4.57 3.98 3.50 4.57 3.98 3.50 OR Quartile 3 3.79 3.85 1.64 3.40 3.64 1.70 3.40 3.64 1.70 p Value 3.3E−4 2.0E−4 0.16 9.8E−4 3.6E−4 0.12 9.8E−4 3.6E−4 0.12 Lower limit of 95% CI 1.83 1.89 0.819 1.64 1.79 0.864 1.64 1.79 0.864 Upper limit of 95% CI 7.84 7.83 3.27 7.04 7.41 3.36 7.04 7.41 3.36 OR Quartile 4 8.89 7.40 2.38 8.26 7.13 2.24 8.26 7.13 2.24 p Value 5.4E−4 3.8E−4 0.028 8.3E−4 4.8E−4 0.040 8.3E−4 4.8E−4 0.040 Lower limit of 95% CI 2.58 2.45 1.10 2.39 2.37 1.04 2.39 2.37 1.04 Upper limit of 95% CI 30.6 22.3 5.14 28.5 21.5 4.81 28.5 21.5 4.81

TABLE 7.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.56 7.04 2.56 7.04 2.52 7.04 Average 9.30 22.6 9.30 22.6 9.21 22.3 Stdev 14.6 31.5 14.6 31.5 14.8 31.1 p (t-test) 0.0023 0.0023 0.0028 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 63 83 63 83 61 85 sCr only Median 2.65 8.16 2.65 8.16 2.61 8.58 Average 9.56 23.3 9.56 23.3 9.45 23.2 Stdev 14.9 32.0 14.9 32.0 15.0 31.8 p (t-test) 0.0016 0.0016 0.0016 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 67 78 67 78 66 79 UO only Median 3.49 7.39 3.49 7.39 3.47 7.04 Average 14.9 22.7 14.9 22.7 15.2 21.7 Stdev 24.2 30.7 24.2 30.7 24.6 30.1 p (t-test) 0.11 0.11 0.19 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 80 48 80 48 77 51 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.65 0.62 0.64 0.65 0.62 0.65 0.65 0.61 SE 0.045 0.045 0.052 0.045 0.045 0.052 0.045 0.045 0.052 p Value 0.0016 0.0010 0.025 0.0016 0.0010 0.025 7.2E−4 5.7E−4 0.031 nCohort Recovered 63 67 80 63 67 80 61 66 77 nCohort Non-recovered 83 78 48 83 78 48 85 79 51 Cutoff Quartile 2 1.90 1.89 1.89 1.90 1.89 1.89 1.90 1.89 1.89 Sensitivity 77% 79% 81% 77% 79% 81% 78% 80% 82% Specificity 29% 30% 29% 29% 30% 29% 30% 30% 30% Cutoff Quartile 3 4.11 4.13 5.38 4.11 4.13 5.38 4.11 4.13 5.38 Sensitivity 59% 60% 60% 59% 60% 60% 60% 61% 61% Specificity 62% 61% 56% 62% 61% 56% 64% 62% 57% Cutoff Quartile 4 19.7 20.0 22.3 19.7 20.0 22.3 19.7 20.0 22.3 Sensitivity 35% 36% 33% 35% 36% 33% 34% 35% 31% Specificity 87% 87% 80% 87% 87% 80% 87% 86% 79% OR Quartile 2 1.35 1.65 1.75 1.35 1.65 1.75 1.45 1.71 1.99 p Value 0.44 0.20 0.21 0.44 0.20 0.21 0.33 0.17 0.12 Lower limit of 95% CI 0.637 0.772 0.731 0.637 0.772 0.731 0.687 0.801 0.833 Upper limit of 95% CI 2.85 3.52 4.18 2.85 3.52 4.18 3.08 3.66 4.74 OR Quartile 3 2.34 2.39 1.96 2.34 2.39 1.96 2.66 2.54 2.07 p Value 0.013 0.011 0.069 0.013 0.011 0.069 0.0048 0.0066 0.048 Lower limit of 95% CI 1.20 1.23 0.948 1.20 1.23 0.948 1.35 1.30 1.01 Upper limit of 95% CI 4.58 4.67 4.06 4.58 4.67 4.06 5.24 4.97 4.25 OR Quartile 4 3.69 3.61 2.00 3.69 3.61 2.00 3.43 3.48 1.74 p Value 0.0032 0.0028 0.095 0.0032 0.0028 0.095 0.0054 0.0037 0.18 Lower limit of 95% CI 1.55 1.56 0.887 1.55 1.56 0.887 1.44 1.50 0.777 Upper limit of 95% CI 8.80 8.37 4.51 8.80 8.37 4.51 8.17 8.06 3.91

TABLE 7.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.79 10.3 2.65 10.0 2.61 10.2 Average 9.37 25.1 9.50 24.1 9.44 23.6 Stdev 15.1 32.8 15.4 32.2 15.7 31.7 p (t-test) 3.4E−4 9.0E−4 0.0014 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 71 71 67 75 64 78 sCr only Median 2.79 10.3 2.72 10.8 2.65 11.3 Average 9.77 25.1 9.67 25.0 9.54 24.9 Stdev 15.5 33.0 15.6 32.8 15.7 32.6 p (t-test) 4.8E−4 4.8E−4 4.6E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 73 69 72 70 71 71 UO only Median 3.49 8.58 3.49 7.04 3.51 7.04 Average 15.9 22.3 15.6 21.9 15.9 21.3 Stdev 26.3 29.6 25.6 30.0 25.8 29.5 p (t-test) 0.22 0.22 0.29 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 74 45 68 51 67 53 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.67 0.64 0.67 0.67 0.63 0.67 0.68 0.62 SE 0.045 0.045 0.053 0.045 0.045 0.052 0.045 0.045 0.052 p Value 2.1E−4 2.7E−4 0.011 1.9E−4 1.5E−4 0.014 1.7E−4 7.0E−5 0.027 nCohort Recovered 71 73 74 67 72 68 64 71 67 nCohort Non-recovered 71 69 45 75 70 51 78 71 53 Cutoff Quartile 2 1.90 1.90 1.88 1.90 1.90 1.88 1.90 1.90 1.89 Sensitivity 77% 78% 82% 79% 79% 84% 78% 79% 83% Specificity 28% 29% 30% 30% 29% 32% 30% 30% 31% Cutoff Quartile 3 4.99 4.99 5.74 4.99 4.99 5.74 4.99 4.99 6.06 Sensitivity 65% 65% 64% 64% 66% 63% 64% 66% 60% Specificity 65% 64% 58% 66% 65% 59% 67% 66% 58% Cutoff Quartile 4 20.7 20.7 22.6 20.7 20.7 22.6 20.7 20.7 22.3 Sensitivity 38% 38% 33% 36% 37% 31% 35% 37% 30% Specificity 87% 86% 80% 87% 86% 79% 86% 86% 79% OR Quartile 2 1.35 1.45 1.96 1.57 1.51 2.57 1.52 1.57 2.23 p Value 0.44 0.34 0.15 0.25 0.29 0.042 0.28 0.25 0.075 Lower limit of 95% CI 0.631 0.677 0.786 0.733 0.703 1.03 0.709 0.730 0.922 Upper limit of 95% CI 2.88 3.12 4.87 3.36 3.24 6.38 3.24 3.37 5.40 OR Quartile 3 3.39 3.39 2.51 3.40 3.60 2.41 3.66 3.84 2.12 p Value 5.2E−4 5.2E−4 0.018 5.1E−4 2.8E−4 0.021 2.7E−4 1.5E−4 0.044 Lower limit of 95% CI 1.70 1.70 1.17 1.71 1.80 1.14 1.82 1.91 1.02 Upper limit of 95% CI 6.74 6.75 5.40 6.78 7.20 5.07 7.34 7.69 4.42 OR Quartile 4 4.23 3.81 1.97 3.62 3.66 1.76 3.24 3.52 1.64 p Value 8.6E−4 0.0015 0.11 0.0028 0.0020 0.18 0.0065 0.0028 0.25 Lower limit of 95% CI 1.81 1.67 0.849 1.56 1.60 0.766 1.39 1.54 0.713 Upper limit of 95% CI 9.87 8.70 4.55 8.45 8.36 4.06 7.53 8.04 3.76

TABLE 7.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 3.03 8.85 3.09 8.58 3.15 8.16 Average 13.3 22.6 13.4 22.2 13.5 21.9 Stdev 22.6 30.6 22.7 30.4 22.8 30.3 p (t-test) 0.035 0.044 0.055 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 93 56 92 57 91 58 sCr only Median 2.91 9.73 2.91 9.73 2.91 9.73 Average 9.79 25.5 9.79 25.5 9.79 25.5 Stdev 15.0 33.7 15.0 33.7 15.0 33.7 p (t-test) 2.0E−4 2.0E−4 2.0E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 83 66 83 66 83 66 UO only Median 3.32 9.46 3.32 9.46 3.47 8.16 Average 14.6 21.7 14.6 21.7 14.8 20.9 Stdev 24.7 28.8 24.7 28.8 24.9 28.5 p (t-test) 0.12 0.12 0.18 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 100 48 100 48 98 50 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.66 0.64 0.64 0.66 0.64 0.64 0.66 0.63 SE 0.048 0.045 0.050 0.048 0.045 0.050 0.047 0.045 0.050 p Value 0.0024 5.1E−4 0.0057 0.0034 5.1E−4 0.0057 0.0044 5.1E−4 0.010 nCohort Recovered 93 83 100 92 83 100 91 83 98 nCohort Non-recovered 56 66 48 57 66 48 58 66 50 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 82% 80% 81% 82% 80% 81% 83% 80% 82% Specificity 29% 29% 28% 29% 29% 28% 30% 29% 29% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 64% 64% 67% 63% 64% 67% 62% 64% 64% Specificity 58% 60% 58% 58% 60% 58% 57% 60% 57% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 36% 38% 35% 35% 38% 35% 34% 38% 34% Specificity 81% 84% 80% 80% 84% 80% 80% 84% 80% OR Quartile 2 1.88 1.66 1.69 1.95 1.66 1.69 2.02 1.66 1.82 p Value 0.13 0.20 0.23 0.11 0.20 0.23 0.090 0.20 0.16 Lower limit of 95% CI 0.831 0.768 0.723 0.863 0.768 0.723 0.895 0.768 0.783 Upper limit of 95% CI 4.26 3.58 3.93 4.42 3.58 3.93 4.58 3.58 4.24 OR Quartile 3 2.49 2.65 2.76 2.33 2.65 2.76 2.18 2.65 2.37 p Value 0.0089 0.0042 0.0057 0.015 0.0042 0.0057 0.023 0.0042 0.016 Lower limit of 95% CI 1.26 1.36 1.34 1.18 1.36 1.34 1.11 1.36 1.17 Upper limit of 95% CI 4.94 5.17 5.67 4.59 5.17 5.67 4.28 5.17 4.79 OR Quartile 4 2.31 3.28 2.19 2.22 3.28 2.19 2.13 3.28 2.01 p Value 0.028 0.0026 0.045 0.037 0.0026 0.045 0.047 0.0026 0.073 Lower limit of 95% CI 1.09 1.52 1.02 1.05 1.52 1.02 1.01 1.52 0.936 Upper limit of 95% CI 4.90 7.11 4.73 4.70 7.11 4.73 4.51 7.11 4.31

Example 8. Use of Insulin-Like Growth Factor-Binding Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 8.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 375 822 316 824 316 824 Average 662 2930 486 2930 486 2930 Stdev 763 5170 555 5160 555 5160 p (t-test) 0.17 0.16 0.16 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 2240 18800 1890 18800 1890 18800 n (Patient) 10 139 9 140 9 140 sCr only Median 463 828 457 826 457 826 Average 1470 3020 1510 3000 1510 3000 Stdev 3810 5200 3900 5180 3900 5180 p (t-test) 0.18 0.20 0.20 Min 30.5 20.6 30.5 20.6 305 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 23 126 22 127 22 127 UO only Median 615 1010 569 1000 567 1010 Average 2050 3570 1900 3540 1640 3680 Stdev 4200 5720 3920 5740 3380 5880 p (t-test) 0.066 0.049 0.014 Min 20.6 172 20.6 172 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 75 73 67 81 64 84 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.63 0.65 0.76 0.63 0.66 0.76 0.63 0.68 SE 0.074 0.059 0.045 0.068 0.060 0.044 0.068 0.060 0.043 p Value 0.0063 0.027 9.2E−4 1.4E−4 0.033 2.6E−4 1.4E−4 0.033 3.9E−5 nCohort Recovered 10 23 75 9 22 67 9 22 64 nCohort Non-recovered 139 126 73 140 127 81 140 127 84 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 77% 76% 85% 77% 76% 85% 77% 76% 86% Specificity 50% 30% 35% 56% 32% 37% 56% 32% 39% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 53% 54% 63% 53% 54% 60% 53% 54% 61% Specificity 80% 70% 63% 89% 68% 63% 89% 68% 64% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 26% 28% 27% 26% 28% 28% 26% 28% 30% Specificity 80% 87% 77% 89% 86% 79% 89% 86% 81% OR Quartile 2 3.34 1.40 2.99 4.22 1.51 3.42 4.22 1.51 3.85 p Value 0.069 0.50 0.0072 0.040 0.41 0.0022 0.040 0.41 8.4E−4 Lower limit of 95% CI 0.910 0.526 1.35 1.07 0.563 1.56 1.07 0.563 1.74 Upper limit of 95% CI 12.3 3.72 6.64 16.6 4.05 7.53 16.6 4.05 8.48 OR Quartile 3 4.42 2.68 2.86 8.97 2.47 2.57 8.97 2.47 2.75 p Value 0.066 0.043 0.0020 0.041 0.066 0.0054 0.041 0.066 0.0031 Lower limit of 95% CI 0.907 1.03 1.47 1.09 0.943 1.32 1.09 0.943 1.41 Upper limit of 95% CI 21.6 6.96 5.57 73.6 6.47 5.01 73.6 6.47 5.40 OR Quartile 4 1.40 2.56 1.29 2.87 2.41 1.50 2.87 2.41 1.84 p Value 0.68 0.15 0.51 0.33 0.18 0.30 0.33 0.18 0.13 Lower limit of 95% CI 0.284 0.717 0.610 0.348 0.671 0.701 0.348 0.671 0.839 Upper limit of 95% CI 6.89 9.17 2.72 23.8 8.65 3.22 23.8 8.65 4.02

TABLE 8.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 416 907 321 907 316 911 Average 578 3250 494 3180 467 3160 Stdev 545 5420 454 5350 448 5330 p (t-test) 0.013 0.020 0.022 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 2240 18800 1890 18800 1890 18800 n (Patient) 26 123 22 127 21 128 sCr only Median 463 911 451 904 442 907 Average 1200 3320 1230 3270 1240 3250 Stdev 3100 5440 3190 5410 3240 5390 p (t-test) 0.030 0.041 0.046 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 35 112 33 114 32 115 UO only Median 626 1070 611 1060 569 1070 Average 2110 3710 1770 3850 1610 3920 Stdev 4330 5830 3610 6070 3440 6040 p (t-test) 0.062 0.014 0.0061 Min 20.6 172 20.6 172 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 81 63 72 72 69 75 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.66 0.66 0.77 0.66 0.68 0.78 0.66 0.70 SE 0.048 0.049 0.046 0.047 0.051 0.045 0.045 0.051 0.043 p Value 1.2E−6 0.0013 4.9E−4 1.3E−8 0.0022 7.0E−5 4.8E−10 0.0013 4.8E−6 nCohort Recovered 26 35 81 22 33 72 21 32 69 nCohort Non-recovered 123 112 63 127 114 72 128 115 75 Cutoff Quartile 2 401 407 399 401 407 399 401 407 399 Sensitivity 80% 78% 89% 80% 78% 89% 80% 78% 89% Specificity 46% 34% 36% 55% 36% 39% 57% 38% 41% Cutoff Quartile 3 794 808 812 794 808 812 794 808 812 Sensitivity 56% 57% 65% 56% 56% 64% 56% 57% 65% Specificity 77% 71% 62% 82% 70% 64% 86% 72% 67% Cutoff Quartile 4 1830 1850 1840 1830 1850 1840 1830 1850 1840 Sensitivity 29% 30% 29% 29% 30% 29% 29% 30% 31% Specificity 92% 91% 78% 95% 91% 79% 95% 91% 81% OR Quartile 2 3.36 1.82 4.46 4.90 2.03 5.09 5.49 2.16 5.72 p Value 0.0074 0.16 0.0012 0.0010 0.096 2.6E−4 5.7E−4 0.073 9.7E−5 Lower limit of 95% CI 1.38 0.794 1.80 1.90 0.881 2.12 2.09 0.931 2.38 Upper limit of 95% CI 8.16 4.15 11.1 12.6 4.70 12.2 14.5 5.01 13.7 OR Quartile 3 4.26 3.33 3.01 5.71 2.94 3.13 7.71 3.32 3.77 p Value 0.0037 0.0042 0.0016 0.0027 0.011 0.0010 0.0016 0.0059 1.7E−4 Lower limit of 95% CI 1.60 1.46 1.52 1.83 1.28 1.59 2.16 1.41 1.89 Upper limit of 95% CI 11.3 7.59 5.96 17.8 6.75 6.18 27.5 7.81 7.52 OR Quartile 4 4.97 4.65 1.40 8.63 4.25 1.56 8.13 4.06 1.91 p Value 0.036 0.016 0.38 0.039 0.024 0.25 0.045 0.029 0.10 Lower limit of 95% CI 1.11 1.33 0.657 1.12 1.21 0.730 1.05 1.16 0.875 Upper limit of 95% CI 22.1 16.2 2.98 66.5 14.9 3.35 62.8 14.2 4.15

TABLE 8.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 451 942 442 962 434 942 Average 863 3470 528 3500 525 3430 Stdev 2000 5620 333 5620 324 5570 p (t-test) 0.0065 0.0025 0.0044 Min 30.5 20.6 30.5 20.6 88.7 20.6 Max 12500 18800 1430 18800 1430 18800 n (Patient) 37 109 34 112 31 115 sCr only Median 540 911 512 929 512 929 Average 1330 3500 1070 3560 1070 3560 Stdev 3220 5620 2800 5640 2800 5640 p (t-test) 0.017 0.0067 0.0067 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 45 100 43 102 43 102 UO only Median 685 1120 637 1120 766 1000 Average 2180 4160 2060 4160 2230 3800 Stdev 4250 6280 4170 6190 4380 5970 p (t-test) 0.036 0.025 0.099 Min 20.6 172 20.6 172 20.6 30.5 Max 18800 18800 18800 18800 18800 18800 n (Patient) 70 58 66 62 59 69 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.65 0.66 0.74 0.67 0.67 0.73 0.67 0.63 SE 0.045 0.047 0.049 0.044 0.047 0.048 0.046 0.047 0.049 p Value 2.5E−6 0.0018 8.6E−4 1.0E−7 3.0E−4 2.6E−4 3.5E−7 3.0E−4 0.0056 nCohort Recovered 37 45 70 34 43 66 31 43 59 nCohort Non-recovered 109 100 58 112 102 62 115 102 69 Cutoff Quartile 2 404 401 414 404 401 414 404 401 414 Sensitivity 80% 78% 90% 79% 78% 89% 79% 78% 87% Specificity 41% 31% 37% 41% 33% 38% 42% 33% 39% Cutoff Quartile 3 790 794 878 790 794 878 790 794 878 Sensitivity 58% 56% 62% 58% 57% 61% 57% 57% 57% Specificity 73% 62% 60% 76% 65% 61% 77% 65% 58% Cutoff Quartile 4 1770 1790 2020 1770 1790 2020 1770 1790 2020 Sensitivity 33% 34% 31% 33% 34% 32% 32% 34% 29% Specificity 97% 93% 80% 100%  95% 82% 100%  95% 80% OR Quartile 2 2.70 1.60 5.12 2.71 1.76 4.79 2.74 1.76 4.26 p Value 0.016 0.24 0.0010 0.018 0.16 9.7E−4 0.019 0.16 0.0012 Lower limit of 95% CI 1.20 0.728 1.93 1.19 0.794 1.89 1.18 0.794 1.78 Upper limit of 95% CI 6.04 3.52 13.6 6.17 3.88 12.1 6.36 3.88 10.2 OR Quartile 3 3.70 2.10 2.45 4.49 2.46 2.44 4.62 2.46 1.77 p Value 0.0018 0.044 0.014 7.8E−4 0.017 0.014 0.0011 0.017 0.11 Lower limit of 95% CI 1.63 1.02 1.20 1.87 1.17 1.20 1.84 1.17 0.876 Upper limit of 95% CI 8.39 4.31 5.01 10.8 5.15 4.96 11.6 5.15 3.57 OR Quartile 4 17.8 7.21 1.80 34.3 10.7 2.14 30.1 10.7 1.60 p Value 0.0054 0.0018 0.15 0.014 0.0017 0.069 0.018 0.0017 0.26 Lower limit of 95% CI 2.34 2.08 0.803 2.04 2.45 0.942 1.79 2.45 0.704 Upper limit of 95% CI 135 25.0 4.04 575 46.9 4.87 505 46.9 3.63

TABLE 8.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 463 1070 427 1050 427 1050 Average 963 3710 967 3580 980 3530 Stdev 2490 5740 2640 5630 2710 5590 p (t-test) 0.0030 0.0068 0.0097 Min 21.1 20.6 21.1 20.6 88.7 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 43 99 38 104 36 106 sCr only Median 526 1060 526 1060 526 1060 Average 1330 3720 1330 3720 1360 3660 Stdev 3420 5700 3420 5700 3480 5660 p (t-test) 0.0075 0.0075 0.011 Min 21.1 20.6 21.1 20.6 30.5 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 50 92 50 92 48 94 UO only Median 747 1070 776 1040 797 992 Average 2530 4040 2760 3660 2890 3500 Stdev 4770 6130 5070 5830 5220 5670 p (t-test) 0.14 0.37 0.55 Min 20.6 172 20.6 30.5 20.6 21.1 Max 18800 18800 18800 18800 18800 18800 n (Patient) 62 57 54 65 50 70 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.68 0.63 0.73 0.68 0.61 0.72 0.68 0.58 SE 0.044 0.045 0.051 0.044 0.045 0.051 0.045 0.046 0.052 p Value 5.5E−7 5.0E−5 0.0092 2.8E−7 5.0E−5 0.030 6.4E−7 1.1E−4 0.13 nCohort Recovered 43 50 62 38 50 54 36 48 50 nCohort Non-recovered 99 92 57 104 92 65 106 94 70 Cutoff Quartile 2 397 397 419 397 397 419 397 397 420 Sensitivity 81% 80% 84% 81% 80% 83% 80% 80% 81% Specificity 40% 36% 34% 42% 36% 35% 42% 35% 34% Cutoff Quartile 3 812 812 901 812 812 901 812 812 904 Sensitivity 62% 61% 60% 61% 61% 57% 60% 61% 54% Specificity 77% 70% 58% 79% 70% 57% 81% 71% 56% Cutoff Quartile 4 1870 1870 2240 1870 1870 2240 1870 1870 2240 Sensitivity 34% 36% 28% 34% 36% 28% 33% 35% 27% Specificity 95% 94% 77% 97% 94% 78% 97% 94% 78% OR Quartile 2 2.75 2.31 2.73 3.05 2.31 2.66 2.89 2.16 2.26 p Value 0.012 0.034 0.026 0.0067 0.034 0.025 0.011 0.051 0.057 Lower limit of 95% CI 1.25 1.07 1.13 1.36 1.07 1.13 1.28 0.996 0.975 Upper limit of 95% CI 6.07 5.01 6.62 6.85 5.01 6.27 6.54 4.71 5.23 OR Quartile 3 5.30 3.63 2.05 5.76 3.63 1.78 6.31 3.74 1.51 p Value 6.1E−5 5.9E−4 0.055 8.5E−5 5.9E−4 0.12 7.6E−5 5.4E−4 0.27 Lower limit of 95% CI 2.34 1.74 0.985 2.41 1.74 0.859 2.53 1.77 0.728 Upper limit of 95% CI 12.0 7.57 4.25 13.8 7.57 3.69 15.7 7.90 3.14 OR Quartile 4 10.7 8.76 1.34 18.8 8.76 1.34 17.3 8.11 1.32 p Value 0.0017 6.2E−4 0.49 0.0046 6.2E−4 0.49 0.0059 9.6E−4 0.52 Lower limit of 95% CI 2.44 2.53 0.584 2.47 2.53 0.578 2.27 2.34 0.564 Upper limit of 95% CI 47.0 30.4 3.07 143 30.4 3.11 131 28.1 3.10

TABLE 8.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 747 942 729 1000 729 1000 Average 2110 3690 2130 3660 2130 3660 Stdev 4460 5630 4480 5600 4480 5600 p (t-test) 0.058 0.066 0.066 Min 20.6 166 20.6 166 20.6 166 Max 18800 18800 18800 18800 18800 18800 n (Patient) 86 63 85 64 85 64 sCr only Median 569 1070 583 1070 583 1070 Average 1430 4010 1470 3920 1470 3920 Stdev 3400 5910 3450 5860 3450 5860 p (t-test) 0.0015 0.0027 0.0027 Min 21.1 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 71 78 69 80 69 80 UO only Median 747 1030 747 1030 682 1030 Average 2200 3910 2230 3790 2210 3760 Stdev 4320 6050 4360 5970 4400 5870 p (t-test) 0.048 0.069 0.070 Min 20.6 175 20.6 172 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 96 52 94 54 92 56 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.62 0.69 0.63 0.62 0.67 0.62 0.62 0.67 0.63 SE 0.047 0.043 0.049 0.047 0.044 0.049 0.047 0.044 0.048 p Value 0.013 8.5E−6 0.011 0.010 1.2E−4 0.018 0.010 1.2E−4 0.0090 nCohort Recovered 86 71 96 85 69 94 85 69 92 nCohort Non-recovered 63 78 52 64 80 54 64 80 56 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 84% 85% 88% 84% 82% 87% 84% 82% 88% Specificity 31% 35% 32% 32% 33% 32% 32% 33% 33% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 56% 62% 60% 56% 60% 59% 56% 60% 61% Specificity 53% 62% 55% 54% 61% 55% 54% 61% 57% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 33% 40% 29% 33% 39% 28% 33% 39% 29% Specificity 80% 90% 77% 80% 90% 77% 80% 90% 77% OR Quartile 2 2.43 2.99 3.66 2.51 2.36 3.15 2.51 2.36 3.39 p Value 0.033 0.0062 0.0076 0.027 0.028 0.013 0.027 0.028 0.0082 Lower limit of 95% CI 1.07 1.36 1.41 1.11 1.10 1.27 1.11 1.10 1.37 Upper limit of 95% CI 5.48 6.55 9.48 5.68 5.06 7.78 5.68 5.06 8.36 OR Quartile 3 1.44 2.61 1.82 1.52 2.33 1.80 1.52 2.33 2.01 p Value 0.28 0.0045 0.087 0.21 0.012 0.089 0.21 0.012 0.043 Lower limit of 95% CI 0.748 1.35 0.918 0.790 1.21 0.914 0.790 1.21 1.02 Upper limit of 95% CI 2.76 5.05 3.61 2.91 4.51 3.55 2.91 4.51 3.95 OR Quartile 4 2.03 6.03 1.36 1.95 5.60 1.26 1.95 5.60 1.35 p Value 0.063 9.5E−5 0.43 0.078 1.8E−4 0.55 0.078 1.8E−4 0.43 Lower limit of 95% CI 0.963 2.45 0.634 0.928 2.27 0.587 0.928 2.27 0.634 Upper limit of 95% CI 4.28 14.9 2.93 4.11 13.8 2.70 4.11 13.8 2.88

Example 9. Use of Insulin-Like Growth Factor-Binding Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 9.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or scrum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 553 904 463 904 448 901 Average 1110 3330 1110 3300 1120 3270 Stdev 2570 5510 2640 5470 2680 5450 p (t-test) 0.019 0.024 0.028 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 15900 18800 15900 18800 15900 18800 n (Patient) 37 112 35 114 34 115 sCr only Median 601 920 601 920 592 916 Average 1400 3460 1400 3460 1410 3440 Stdev 3380 5560 3380 5560 3410 5540 p (t-test) 0.018 0.018 0.021 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 49 100 49 100 48 101 UO only Median 637 1130 630 1120 626 1070 Average 2300 3660 2380 3410 2230 3570 Stdev 4630 5640 4770 5400 4510 5630 p (t-test) 0.11 0.23 0.11 Min 20.6 172 20.6 172 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 94 54 88 60 85 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.65 0.63 0.66 0.66 0.63 0.66 0.66 0.63 0.67 SE 0.049 0.047 0.048 0.049 0.047 0.046 0.049 0.047 0.046 p Value 0.0022 0.0050 0.0012 0.0012 0.0050 7.3E−4 9.1E−4 0.0044 2.9E−4 nCohort Recovered 37 49 94 35 49 88 34 48 85 nCohort Non-recovered 112 100 54 114 100 60 115 101 63 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 79% 77% 89% 79% 77% 90% 79% 77% 90% Specificity 35% 29% 33% 37% 29% 35% 38% 29% 36% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 54% 56% 65% 54% 56% 63% 55% 56% 63% Specificity 62% 61% 59% 63% 61% 59% 65% 62% 60% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 30% 33% 31% 30% 33% 30% 30% 33% 30% Specificity 89% 90% 79% 89% 90% 78% 88% 90% 79% OR Quartile 2 1.99 1.34 3.94 2.22 1.34 4.89 2.35 1.40 5.45 p Value 0.098 0.46 0.0048 0.057 0.46 0.0011 0.043 0.40 4.7E−4 Lower limit of 95% CI 0.882 0.617 1.52 0.976 0.617 1.89 1.03 0.642 2.11 Upper limit of 95% CI 4.47 2.91 10.2 5.03 2.91 12.7 5.36 3.04 14.1 OR Quartile 3 1.96 2.01 2.60 2.02 2.01 2.49 2.22 2.16 2.61 p Value 0.082 0.050 0.0069 0.077 0.050 0.0080 0.049 0.032 0.0052 Lower limit of 95% CI 0.918 1.00 1.30 0.926 1.00 1.27 1.00 1.07 1.33 Upper limit of 95% CI 4.21 4.04 5.20 4.39 4.04 4.90 4.91 4.37 5.11 OR Quartile 4 3.60 4.33 1.70 3.29 4.33 1.56 3.15 4.17 1.61 p Value 0.024 0.0046 0.17 0.036 0.0046 0.25 0.044 0.0058 0.21 Lower limit of 95% CI 1.18 1.57 0.797 1.08 1.57 0.735 1.03 1.51 0.760 Upper limit of 95% CI 10.9 12.0 3.63 10.1 12.0 3.30 9.62 11.5 3.40

TABLE 9.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 553 992 540 992 540 992 Average 1290 3560 1290 3520 1290 3520 Stdev 3110 5650 3170 5600 3170 5600 p (t-test) 0.0083 0.011 0.011 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 51 98 49 100 49 100 sCr only Median 576 1070 569 1020 569 1020 Average 1570 3580 1590 3550 1590 3550 Stdev 3770 5590 3810 5570 3810 5570 p (t-test) 0.019 0.022 0.022 Min 30.5 20.6 30.5 20.6 30.5 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 56 91 55 92 55 92 UO only Median 672 1190 640 1180 640 1180 Average 2280 3800 2160 3870 2160 3870 Stdev 4600 5810 4380 5950 4380 5950 p (t-test) 0.088 0.049 0.049 Min 20.6 172 20.6 172 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 94 50 89 55 89 55 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.65 0.66 0.68 0.65 0.67 0.68 0.65 0.67 SE 0.045 0.045 0.049 0.044 0.045 0.047 0.044 0.045 0.047 p Value 1.1E−4 8.6E−4 8.9E−4 3.3E−5 7.7E−4 2.7E−4 3.3E−5 7.7E−4 2.7E−4 nCohort Recovered 51 56 94 49 55 89 49 55 89 nCohort Non-recovered 98 91 50 100 92 55 100 92 55 Cutoff Quartile 2 401 407 399 401 407 399 401 407 399 Sensitivity 81% 79% 90% 81% 79% 91% 81% 79% 91% Specificity 35% 32% 33% 37% 33% 35% 37% 33% 35% Cutoff Quartile 3 794 808 812 794 808 812 794 808 812 Sensitivity 59% 59% 68% 60% 60% 67% 60% 60% 67% Specificity 67% 64% 60% 69% 65% 61% 69% 65% 61% Cutoff Quartile 4 1830 1850 1840 1830 1850 1840 1830 1850 1840 Sensitivity 34% 34% 32% 33% 34% 31% 33% 34% 31% Specificity 90% 89% 79% 90% 89% 79% 90% 89% 79% OR Quartile 2 2.27 1.80 4.43 2.48 1.87 5.34 2.48 1.87 5.34 p Value 0.035 0.13 0.0042 0.020 0.11 0.0012 0.020 0.11 0.0012 Lower limit of 95% CI 1.06 0.844 1.60 1.15 0.877 1.93 1.15 0.877 1.93 Upper limit of 95% CI 4.86 3.82 12.3 5.33 3.98 14.8 5.33 3.98 14.8 OR Quartile 3 2.90 2.63 3.13 3.40 2.82 3.17 3.40 2.82 3.17 p Value 0.0032 0.0060 0.0020 9.8E−4 0.0035 0.0013 9.8E−4 0.0035 0.0013 Lower limit of 95% CI 1.43 1.32 1.52 1.64 1.41 1.57 1.64 1.41 1.57 Upper limit of 95% CI 5.89 5.23 6.45 7.04 5.64 6.42 7.04 5.64 6.42 OR Quartile 4 4.67 4.31 1.74 4.33 4.15 1.65 4.33 4.15 1.65 p Value 0.0029 0.0026 0.16 0.0046 0.0034 0.20 0.0046 0.0034 0.20 Lower limit of 95% CI 1.70 1.66 0.804 1.57 1.60 0.768 1.57 1.60 0.768 Upper limit of 95% CI 12.9 11.1 3.77 12.0 10.7 3.54 12.0 10.7 3.54

TABLE 9.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 569 1070 569 1070 569 1070 Average 1430 3860 1430 3860 1270 3920 Stdev 3210 5940 3210 5940 2920 5960 p (t-test) 0.0039 0.0039 0.0017 Min 21.1 20.6 21.1 20.6 21.1 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 63 83 63 83 61 85 sCr only Median 601 1020 601 1020 592 1070 Average 1680 3810 1680 3810 1520 3920 Stdev 3760 5850 3760 5850 3540 5900 p (t-test) 0.012 0.012 0.0044 Min 21.1 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 67 78 67 78 66 79 UO only Median 722 1200 722 1200 766 1070 Average 2260 4430 2260 4430 2340 4190 Stdev 4410 6420 4410 6420 4480 6300 p (t-test) 0.026 0.026 0.054 Min 20.6 194 20.6 194 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 80 48 80 48 77 51 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.65 0.67 0.68 0.65 0.67 0.68 0.66 0.64 SE 0.044 0.045 0.051 0.044 0.045 0.051 0.044 0.045 0.051 p Value 4.2E−5 6.6E−4 9.0E−4 4.2E−5 6.6E−4 9.0E−4 3.0E−5 2.2E−4 0.0076 nCohort Recovered 63 67 80 63 67 80 61 66 77 nCohort Non-recovered 83 78 48 83 78 48 85 79 51 Cutoff Quartile 2 404 401 414 404 401 414 404 401 414 Sensitivity 82% 81% 92% 82% 81% 92% 81% 81% 88% Specificity 35% 31% 35% 35% 31% 35% 34% 32% 34% Cutoff Quartile 3 790 794 878 790 794 878 790 794 878 Sensitivity 61% 60% 65% 61% 60% 65% 61% 61% 61% Specificity 65% 61% 59% 65% 61% 59% 66% 62% 57% Cutoff Quartile 4 1770 1790 2020 1770 1790 2020 1770 1790 2020 Sensitivity 36% 37% 31% 36% 37% 31% 36% 38% 29% Specificity 89% 88% 79% 89% 88% 79% 90% 89% 78% OR Quartile 2 2.43 1.92 5.92 2.43 1.92 5.92 2.26 1.99 3.82 p Value 0.022 0.095 0.0019 0.022 0.095 0.0019 0.035 0.077 0.0070 Lower limit of 95% CI 1.14 0.893 1.93 1.14 0.893 1.93 1.06 0.927 1.44 Upper limit of 95% CI 5.21 4.12 18.2 5.21 4.12 18.2 4.83 4.28 10.1 OR Quartile 3 2.97 2.39 2.60 2.97 2.39 2.60 3.00 2.54 2.07 p Value 0.0017 0.011 0.012 0.0017 0.011 0.012 0.0017 0.0066 0.048 Lower limit of 95% CI 1.50 1.23 1.24 1.50 1.23 1.24 1.51 1.30 1.01 Upper limit of 95% CI 5.87 4.67 5.45 5.87 4.67 5.45 5.95 4.97 4.25 OR Quartile 4 4.53 4.36 1.68 4.53 4.36 1.68 5.26 5.16 1.47 p Value 0.0011 9.0E−4 0.21 0.0011 9.0E−4 0.21 6.2E−4 3.8E−4 0.35 Lower limit of 95% CI 1.83 1.83 0.748 1.83 1.83 0.748 2.03 2.09 0.656 Upper limit of 95% CI 11.2 10.4 3.79 11.2 10.4 3.79 13.6 12.8 3.30

TABLE 9.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 583 1250 583 1220 573 1240 Average 1360 4400 1230 4360 1220 4240 Stdev 3030 6260 2790 6210 2850 6120 p (t-test) 3.3E−4 2.2E−4 4.0E−4 Min 21.1 20.6 21.1 20.6 21.1 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 71 71 67 75 64 78 sCr only Median 601 1180 592 1210 583 1250 Average 1600 4230 1450 4350 1440 4320 Stdev 3620 6100 3400 6140 3420 6100 p (t-test) 0.0020 6.3E−4 6.9E−4 Min 21.1 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 73 69 72 70 71 71 UO only Median 776 1180 797 1070 808 1070 Average 2680 4200 2600 4120 2650 4000 Stdev 4940 6240 4730 6310 4760 6220 p (t-test) 0.14 0.14 0.18 Min 20.6 194 20.6 172 20.6 169 Max 18800 18800 18800 18800 18800 18800 n (Patient) 74 45 68 51 67 53 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.69 0.64 0.70 0.70 0.61 0.70 0.71 0.60 SE 0.043 0.045 0.053 0.043 0.044 0.053 0.043 0.043 0.052 p Value 1.7E−6 2.4E−5 0.0087 3.3E−6 5.7E−6 0.033 2.1E−6 1.9E−6 0.064 nCohort Recovered 71 73 74 67 72 68 64 71 67 nCohort Non-recovered 71 69 45 75 70 51 78 71 53 Cutoff Quartile 2 397 397 419 397 397 419 397 397 420 Sensitivity 85% 84% 89% 83% 84% 84% 82% 85% 83% Specificity 35% 34% 34% 34% 35% 32% 34% 35% 31% Cutoff Quartile 3 812 812 901 812 812 901 812 812 904 Sensitivity 65% 64% 62% 64% 64% 59% 64% 65% 58% Specificity 65% 63% 57% 66% 64% 56% 67% 65% 57% Cutoff Quartile 4 1870 1870 220 1870 1870 2240 1870 1870 2240 Sensitivity 39% 39% 29% 39% 40% 27% 38% 41% 26% Specificity 89% 88% 77% 90% 89% 76% 91% 90% 76% OR Quartile 2 2.96 2.75 4.08 2.49 2.85 2.57 2.39 2.96 2.23 p Value 0.0083 0.014 0.0085 0.022 0.011 0.042 0.027 0.0083 0.075 Lower limit of 95% CI 1.32 1.23 1.43 1.14 1.27 1.03 1.10 1.32 0.922 Upper limit of 95% CI 6.64 6.15 11.6 5.45 6.39 6.38 5.20 6.64 5.40 OR Quartile 3 3.39 3.00 2.16 3.40 3.18 1.81 3.66 3.39 1.85 p Value 5.2E−4 0.0016 0.046 5.1E−4 9.3E−4 0.11 2.7E−4 5.2E−4 0.099 Lower limit of 95% CI 1.70 1.51 1.01 1.71 1.60 0.868 1.82 1.70 0.890 Upper limit of 95% CI 6.74 5.94 4.61 6.78 6.32 3.77 7.34 6.74 3.83 OR Quartile 4 5.13 4.57 1.36 5.40 5.33 1.23 6.04 6.31 1.14 p Value 2.6E−4 4.5E−4 0.47 2.8E−4 1.8E−4 0.63 2.3E−4 7.6E−5 0.75 Lower limit of 95% CI 2.14 1.96 0.587 2.17 2.22 0.535 2.32 2.53 0.499 Upper limit of 95% CI 12.3 10.7 3.16 13.4 12.8 2.83 15.7 15.7 2.62

TABLE 9.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 642 1070 686 1070 642 1070 Average 2060 3980 2080 3920 2090 3870 Stdev 4310 5890 4330 5860 4360 5820 p (t-test) 0.023 0.030 0.035 Min 20.6 166 20.6 166 20.6 166 Max 18800 18800 18800 18800 18800 18800 n (Patient) 93 56 92 57 91 58 sCr only Median 622 1070 622 1070 622 1070 Average 1480 4420 1480 4420 1480 4420 Stdev 3210 6310 3210 6310 3210 6310 p (t-test) 3.1E−4 3.1E−4 3.1E−4 Min 21.1 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 83 66 83 66 83 66 UO only Median 747 1070 747 1070 747 1070 Average 2140 4170 2140 4170 2170 4040 Stdev 4250 6230 4250 6230 4280 6140 p (t-test) 0.021 0.021 0.033 Min 20.6 175 20.6 175 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 100 48 100 48 98 50 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.68 0.63 0.62 0.68 0.63 0.63 0.68 0.62 SE 0.048 0.045 0.050 0.048 0.045 0.050 0.048 0.045 0.050 p Value 0.0045 8.8E−5 0.0077 0.011 8.8E−5 0.0077 0.0080 8.8E−5 0.014 nCohort Recovered 93 83 100 92 83 100 91 83 98 nCohort Non-recovered 56 66 48 57 66 48 58 66 50 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 86% 85% 88% 84% 85% 88% 84% 85% 86% Specificity 31% 33% 31% 30% 33% 31% 31% 33% 31% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 59% 62% 60% 58% 62% 60% 59% 62% 60% Specificity 55% 59% 55% 54% 59% 55% 55% 59% 55% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 34% 38% 31% 33% 38% 31% 33% 38% 30% Specificity 80% 84% 78% 79% 84% 78% 79% 84% 78% OR Quartile 2 2.72 2.70 3.14 2.33 2.70 3.14 2.42 2.70 2.71 p Value 0.024 0.017 0.019 0.048 0.017 0.019 0.039 0.017 0.031 Lower limit of 95% CI 1.14 1.20 1.21 1.01 1.20 1.21 1.05 1.20 1.09 Upper limit of 95% CI 6.47 6.10 8.17 5.40 6.10 8.17 5.60 6.10 6.71 OR Quartile 3 1.74 2.36 1.87 1.64 2.36 1.87 1.73 2.36 1.84 p Value 0.10 0.011 0.081 0.15 0.011 0.081 0.11 0.011 0.084 Lower limit of 95% CI 0.891 1.22 0.926 0.840 1.22 0.926 0.888 1.22 0.922 Upper limit of 95% CI 3.41 4.58 3.76 3.19 4.58 3.76 3.36 4.58 3.68 OR Quartile 4 2.00 3.28 1.61 1.92 3.28 1.61 1.85 3.2 1.48 p Value 0.069 0.0026 0.23 0.087 0.0026 0.23 0.11 0.0026 0.32 Lower limit of 95% CI 0.946 1.52 0.744 0.910 1.52 0.744 0.876 1.52 0.686 Upper limit of 95% CI 4.23 7.11 3.49 4.05 7.11 3.49 3.89 7.11 3.19

Example 10. Use of Insulin-Like Growth Factor-Binding Protein 4 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) arc enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 4 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 10.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000286 0.394 0.000286 0.341 0.000286 0.341 Average 0.236 8.15 0.262 8.09 0.262 8.09 Stdev 0.746 19.5 0.786 19.4 0.786 19.4 p (t-test) 0.20 0.23 0.23 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 2.36 120 2.36 120 2.36 120 n (Patient) 10 139 9 140 9 140 sCr only Median 0.000339 0.538 0.000339 0.443 0.000339 0.443 Average 0.740 8.87 0.773 8.80 0.773 8.80 Stdev 2.66 20.3 2.72 20.3 2.72 20.3 p (t-test) 0.058 0.066 0.066 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 12.7 120 12.7 120 12.7 120 n (Patient) 23 126 22 127 22 127 UO only Median 0.126 0.394 0.165 0.265 0.142 0.330 Average 7.26 8.08 7.17 8.07 4.67 9.95 Stdev 19.8 18.3 19.7 18.5 12.3 22.6 p (t-test) 0.79 0.77 0.094 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 110 120 110 73.0 120 n (Patient) 75 73 67 81 64 84 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.86 0.75 0.54 0.87 0.75 0.55 0.87 0.75 0.57 SE 0.045 0.048 0.047 0.042 0.049 0.047 0.042 0.049 0.047 p Value 2.2E−15 1.0E−7 0.35 0 2.7E−7 0.28 0 2.7E−7 0.15 nCohort Recovered 10 23 75 9 22 67 9 22 64 nCohort Non-recovered 139 126 73 140 127 81 140 127 84 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 85% 85% 84% 85% 85% 85% 85% 85% 85% Specificity 70% 39% 20% 78% 41% 22% 78% 41% 22% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 53% 55% 52% 53% 54% 51% 53% 54% 51% Specificity 90% 74% 52% 89% 73% 51% 89% 73% 52% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 27% 29% 30% 27% 29% 28% 27% 29% 30% Specificity 100%  96% 80% 100%  95% 79% 100%  95% 81% OR Quartile 2 13.1 3.62 1.27 19.8 3.94 1.66 19.8 3.94 1.53 p Value 4.2E−4 0.0093 0.58 3.5E−4 0.0061 0.24 3.5E−4 0.0061 0.32 Lower limit of 95% CI 3.14 1.37 0.549 3.85 1.48 0.716 3.85 1.48 0.662 Upper limit of 95% CI 54.8 9.54 2.94 102 10.5 3.84 102 10.5 3.53 OR Quartile 3 10.2 3.43 1.18 8.97 3.17 1.06 8.97 3.17 1.12 p Value 0.029 0.015 0.62 0.041 0.024 0.87 0.041 0.024 0.74 Lower limit of 95% CI 1.26 1.27 0.617 1.09 1.17 0.553 1.09 1.17 0.582 Upper limit of 95% CI 83.1 9.27 2.24 73.6 8.63 2.02 73.6 8.63 2.14 OR Quartile 4 7.97 9.15 1.73 7.14 8.63 1.50 7.14 8.63 1.84 p Value 0.16 0.033 0.16 0.18 0.039 0.30 0.18 0.039 0.13 Lower limit of 95% CI 0.456 1.19 0.811 0.405 1.12 0.701 0.405 1.12 0.839 Upper limit of 95% CI 139 70.4 3.67 126 66.5 3.22 126 66.5 4.02

TABLE 10.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000457 0.633 0.000391 0.449 0.000339 0.446 Average 0.209 9.18 0.234 8.89 0.224 8.83 Stdev 0.501 20.5 0.542 20.2 0.553 20.2 p (t-test) 0.028 0.047 0.053 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 2.36 120 2.36 120 2.36 120 n (Patient) 26 123 22 127 21 128 sCr only Median 0.000471 0.752 0.000471 0.685 0.000471 0.659 Average 0.550 9.94 0.580 9.77 0.584 9.69 Stdev 2.16 21.3 2.23 21.2 2.26 21.1 p (t-test) 0.010 0.014 0.016 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 12.7 120 12.7 120 12.7 120 n (Patient) 35 112 33 114 32 115 UO only Median 0.118 0.555 0.102 0.422 0.0851 0.449 Average 6.50 9.12 4.74 10.6 3.86 11.1 Stdev 18.9 19.5 13.0 23.4 11.3 23.7 p (t-test) 0.42 0.067 0.022 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 110 73.0 120 73.0 120 n (Patient) 81 63 72 72 69 75 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.74 0.58 0.75 0.73 0.59 0.76 0.74 0.60 SE 0.046 0.044 0.048 0.048 0.045 0.047 0.048 0.044 0.047 p Value 1.1E−7 7.2E−8 0.099 1.7E−7 1.3E−7 0.067 2.6E−8 4.8E−8 0.033 nCohort Recovered 26 35 81 22 33 72 21 32 69 nCohort Non-recovered 123 112 63 127 114 72 128 115 75 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 85% 85% 84% 84% 85% 85% 84% 85% 84% Specificity 35% 29% 21% 36% 30% 22% 38% 31% 22% Cutoff Quartile 3 0.170 0.170 0.218 0.170 0.170 0.218 0.170 0.170 0.218 Sensitivity 56% 58% 56% 55% 57% 53% 55% 57% 53% Specificity 77% 74% 54% 77% 73% 53% 81% 75% 54% Cutoff Quartile 4 2.99 3.02 3.01 2.99 3.02 3.01 2.99 3.02 3.01 Sensitivity 31% 32% 32% 30% 32% 32% 30% 31% 33% Specificity 100%  97% 80% 100%  97% 82% 100%  97% 84% OR Quartile 2 2.90 2.24 1.41 3.06 2.48 1.58 3.32 2.62 1.46 p Value 0.027 0.079 0.44 0.027 0.049 0.29 0.019 0.038 0.38 Lower limit of 95% CI 1.13 0.912 0.595 1.13 1.00 0.678 1.22 1.06 0.629 Upper limit of 95% CI 7.45 5.48 3.33 8.24 6.12 3.70 9.05 6.49 3.38 OR Quartile 3 4.26 4.00 1.49 4.18 3.54 1.25 5.29 4.04 1.32 p Value 0.0037 0.0013 0.24 0.0080 0.0036 0.51 0.0043 0.0019 0.40 Lower limit of 95% CI 1.60 1.71 0.767 1.45 1.51 0.649 1.69 1.67 0.686 Upper limit of 95% CI 11.3 9.31 2.88 12.0 8.29 2.40 16.6 9.76 2.55 OR Quartile 4 23.9 16.1 1.89 19.4 14.8 2.13 18.3 14.1 2.64 p Value 0.028 0.0072 0.10 0.040 0.0093 0.057 0.044 0.011 0.018 Lower limit of 95% CI 1.42 2.12 0.882 1.14 1.94 0.978 1.08 1.86 1.18 Upper limit of 95% CI 402 122 4.05 327 112 4.64 310 108 5.89

TABLE 10.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000443 0.710 0.000339 0.685 0.000443 0.633 Average 0.555 10.2 0.201 10.0 0.220 9.78 Stdev 2.20 21.6 0.497 21.3 0.517 21.1 p (t-test) 0.0076 0.0081 0.013 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 13.2 120 2.56 120 2.56 120 n (Patient) 37 109 34 112 31 115 sCr only Median 0.000471 0.866 0.000443 0.866 0.000443 0.866 Average 0.750 11.0 0.467 10.9 0.467 10.9 Stdev 2.70 22.3 1.96 22.1 1.96 22.1 p (t-test) 0.0027 0.0025 0.0025 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 13.2 120 12.7 120 12.7 120 n (Patient) 45 100 43 102 43 102 UO only Median 0.341 0.706 0.341 0.706 0.404 0.265 Average 7.43 9.90 6.75 10.5 7.51 9.44 Stdev 19.3 21.1 18.6 21.5 19.6 20.6 p (t-test) 0.49 0.30 0.59 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 70 58 66 62 59 69 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.76 0.57 0.78 0.78 0.58 0.74 0.78 0.54 SE 0.042 0.040 0.051 0.040 0.038 0.050 0.045 0.038 0.051 p Value 1.9E−9 4.0E−11 0.15 4.8E−12 1.5E−13 0.10 5.4E−8 1.5E−13 0.42 nCohort Recovered 37 45 70 34 43 66 31 43 59 nCohort Non-recovered 109 100 58 112 102 62 115 102 69 Cutoff Quartile 2 0.000339 0.000339 0.000471 0.000339 0.000339 0.000471 0.000339 0.000339 0.000471 Sensitivity 85% 87% 81% 86% 87% 81% 83% 87% 78% Specificity 30% 31% 29% 32% 33% 29% 26% 33% 27% Cutoff Quartile 3 0.168 0.165 0.399 0.168 0.165 0.399 0.168 0.165 0.399 Sensitivity 59% 62% 52% 59% 63% 52% 57% 63% 49% Specificity 76% 73% 51% 79% 77% 52% 77% 77% 49% Cutoff Quartile 4 3.04 3.05 4.89 3.04 3.05 4.89 3.04 3.05 4.89 Sensitivity 33% 35% 29% 33% 35% 31% 32% 35% 28% Specificity 97% 96% 79% 100%  98% 80% 100%  98% 78% OR Quartile 2 2.46 3.02 1.71 2.87 3.31 1.68 1.76 3.31 1.34 p Value 0.046 0.012 0.21 0.021 0.0067 0.22 0.24 0.0067 0.48 Lower limit of 95% CI 1.02 1.28 0.740 1.18 1.39 0.738 0.684 1.39 0.596 Upper limit of 95% CI 5.94 7.14 3.95 7.00 7.84 3.84 4.51 7.84 3.01 OR Quartile 3 4.42 4.49 1.13 5.53 5.56 1.13 4.62 5.56 0.939 p Value 5.4E−4 1.4E−4  0.72 2.4E−4  3.6E−5  0.72 0.0011 3.6E−5  0.86 Lower limit of 95% CI 1.91 2.07 0.565 2.22 2.46 0.566 1.84 2.46 0.469 Upper limit of 95% CI 10.3 9.73 2.28 13.8 12.5 2.27 11.6 12.5 1.88 OR Quartile 4 17.8 11.6 1.52 34.3 22.9 1.80 30.1 22.9 1.34 p Value 0.0054 0.0011 0.31 0.014 0.0024 0.16 0.018 0.0024 0.47 Lower limit of 95% CI 2.34 2.65 0.681 2.04 3.03 0.800 1.79 3.03 0.597 Upper limit of 95% CI 135 50.7 3.40 575 173 4.06 505 173 3.03

TABLE 10.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000471 0.857 0.000471 0.646 0.000471 0.646 Average 1.29 10.9 1.46 10.3 1.52 10.1 Stdev 5.58 22.3 5.93 21.9 6.09 21.7 p (t-test) 0.0063 0.015 0.020 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 35.9 120 35.9 120 35.9 120 n (Patient) 43 99 38 104 36 106 sCr only Median 0.000472 0.869 0.000472 0.869 0.000472 0.866 Average 1.38 11.5 1.38 11.5 1.42 11.3 Stdev 5.44 22.9 5.44 22.9 5.55 22.8 p (t-test) 0.0025 0.0025 0.0036 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 35.9 120 35.9 120 35.9 120 n (Patient) 50 92 50 92 48 94 UO only Median 0.142 0.866 0.399 0.555 0.446 0.330 Average 7.83 10.1 8.92 8.96 9.71 8.45 Stdev 20.4 21.3 21.6 20.1 22.3 19.5 p (t-test) 0.54 0.99 0.74 Min 0.000212 0.000286 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 62 57 54 65 50 70 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.74 0.59 0.70 0.74 0.54 0.70 0.73 0.52 SE 0.042 0.041 0.052 0.046 0.041 0.053 0.047 0.042 0.054 p Value 9.4E−9 1.1E−8 0.071 9.8E−6 1.1E−8 0.40 1.6E−5 2.3E−8 0.73 nCohort Recovered 43 50 62 38 50 54 36 48 50 nCohort Non-recovered 99 92 57 104 92 65 106 94 70 Cutoff Quartile 2 0.000365 0.000365 0.000471 0.000365 0.000365 0.000471 0.000365 0.000365 0.000471 Sensitivity 83% 83% 82% 81% 83% 80% 80% 82% 79% Specificity 44% 40% 29% 42% 40% 28% 42% 40% 26% Cutoff Quartile 3 0.205 0.205 0.404 0.205 0.205 0.404 0.205 0.205 0.424 Sensitivity 61% 63% 54% 58% 63% 51% 58% 63% 49% Specificity 74% 74% 53% 71% 74% 50% 72% 75% 48% Cutoff Quartile 4 3.58 3.58 4.63 3.58 3.58 4.63 3.58 3.58 4.89 Sensitivity 34% 36% 32% 33% 36% 28% 32% 35% 26% Specificity 95% 94% 81% 95% 94% 78% 94% 94% 76% OR Quartile 2 3.82 3.17 1.92 3.05 3.17 1.54 2.89 2.97 1.29 p Value 9.8E−4 0.0038 0.14 0.0067 0.0038 0.32 0.011 0.0064 0.56 Lower limit of 95% CI 1.72 1.45 0.801 1.36 1.45 0.657 1.28 1.36 0.550 Upper limit of 95% CI 8.47 6.92 4.62 6.85 6.92 3.60 6.54 6.48 3.02 OR Quartile 3 4.48 4.86 1.36 3.35 4.86 1.03 3.52 5.06 0.872 p Value 2.2E−4 4.7E−5 0.41 0.0031 4.7E−5 0.93 0.0028 4.2E−5 0.71 Lower limit of 95% CI 2.02 2.27 0.659 1.50 2.27 0.501 1.54 2.33 0.422 Upper limit of 95% CI 9.91 10.4 2.79 7.46 10.4 2.12 8.04 11.0 1.80 OR Quartile 4 10.7 8.76 1.92 8.74 8.76 1.34 8.03 8.11 1.10 p Value 0.0017 6.2E−4 0.13 0.0041 6.2E−4 0.49 0.0059 9.6E−4 0.83 Lower limit of 95% CI 2.44 2.53 0.829 1.99 2.53 0.578 1.82 2.34 0.472 Upper limit of 95% CI 47.0 30.4 4.46 38.5 30.4 3.11 35.4 28.1 2.54

TABLE 10.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0328 0.872 0.0328 0.869 0.0328 0.869 Average 6.80 8.72 6.88 8.59 6.88 8.59 Stdev 21.2 15.5 21.3 15.4 21.3 15.4 p (t-test) 0.54 0.59 0.59 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 59.1 120 59.1 120 59.1 n (Patient) 86 63 85 64 85 64 sCr only Median 0.000473 1.17 0.000473 1.13 0.000473 1.13 Average 2.89 11.9 2.97 11.6 2.97 11.6 Stdev 13.8 21.9 13.9 21.7 13.9 21.7 p (t-test) 0.0033 0.0051 0.0051 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 71 78 69 80 69 80 UO only Median 0.0873 0.899 0.104 0.869 0.0873 0.869 Average 7.22 8.48 7.38 8.17 7.44 8.04 Stdev 20.9 14.9 21.1 14.7 21.4 14.4 p (t-test) 0.70 0.81 0.85 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 50.9 120 50.9 120 50.9 n (Patient) 96 52 94 54 92 56 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.66 0.73 0.60 0.66 0.72 0.59 0.66 0.72 0.60 SE 0.046 0.041 0.050 0.046 0.041 0.049 0.046 0.041 0.049 p Value 4.0E−4 1.6E−8 0.040 6.0E−4 9.2E−8 0.069 6.0E−4 9.2E−8 0.046 nCohort Recovered 86 71 96 85 69 94 85 69 92 nCohort Non-recovered 63 78 52 64 80 54 64 80 56 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 89% 87% 85% 89% 88% 85% 89% 88% 86% Specificity 24% 25% 20% 25% 26% 20% 25% 26% 21% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 65% 67% 58% 64% 65% 56% 64% 65% 55% Specificity 60% 68% 54% 60% 67% 53% 60% 67% 53% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 33% 40% 35% 33% 39% 33% 33% 39% 34% Specificity 80% 90% 80% 80% 90% 80% 80% 90% 80% OR Quartile 2 2.58 2.31 1.36 2.67 2.47 1.46 2.67 2.47 1.56 p Value 0.045 0.054 0.51 0.038 0.038 0.41 0.038 0.038 0.33 Lower limit of 95% CI 1.02 0.985 0.549 1.06 1.05 0.590 1.06 1.05 0.633 Upper limit of 95% CI 6.53 5.42 3.36 6.75 5.80 3.60 6.75 5.80 3.85 OR Quartile 3 2.85 4.17 1.61 2.67 3.71 1.42 2.67 3.71 1.41 p Value 0.0023 4.3E−5 0.17 0.0040 1.5E−4 0.31 0.0040 1.5E−4 0.31 Lower limit of 95% CI 1.45 2.10 0.816 1.37 1.88 0.725 1.37 1.88 0.725 Upper limit of 95% CI 5.60 8.28 3.18 5.23 7.33 2.78 5.23 7.33 2.75 OR Quartile 4 2.03 6.03 2.15 1.95 5.60 1.97 1.95 5.60 2.11 p Value 0.063 9.5E−5 0.049 0.078 1.8E−4 0.078 0.078 1.8E−4 0.053 Lower limit of 95% CI 0.963 2.45 1.00 0.928 2.27 0.925 0.928 2.27 0.991 Upper limit of 95% CI 4.28 14.9 4.59 4.11 13.8 4.21 4.11 13.8 4.50

Example 11. Use of Insulin-Like Growth Factor-Binding Protein 4 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 4 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 11.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by scrum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000471 0.685 0.000471 0.646 0.000471 0.633 Average 3.59 8.94 3.78 8.79 3.89 8.71 Stdev 18.1 19.1 18.6 19.0 18.9 18.9 p (t-test) 0.14 0.17 0.19 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 37 112 35 114 34 115 sCr only Median 0.000471 0.866 0.000471 0.866 0.000472 0.866 Average 3.00 9.87 3.00 9.87 3.07 9.78 Stdev 15.8 20.0 15.8 20.0 16.0 19.9 p (t-test) 0.037 0.037 0.043 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 49 100 49 100 48 101 UO only Median 0.122 0.679 0.122 0.594 0.118 0.633 Average 7.32 8.27 7.73 7.57 6.55 9.17 Stdev 20.8 15.4 21.5 14.8 18.0 20.3 p (t-test) 0.77 0.96 0.41 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 59.1 120 59.1 110 120 n (Patient) 94 54 88 60 85 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.73 0.57 0.71 0.73 0.57 0.69 0.72 0.58 SE 0.046 0.041 0.049 0.046 0.041 0.048 0.047 0.042 0.048 p Value 7.5E−6 3.3E−8 0.15 8.6E−6 3.3E−8 0.15 4.5E−5 2.4E−7 0.087 nCohort Recovered 37 49 94 35 49 88 34 48 85 nCohort Non-recovered 112 100 54 114 100 60 115 101 63 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 85% 87% 83% 85% 87% 85% 84% 86% 86% Specificity 30% 31% 19% 31% 31% 20% 29% 29% 21% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 57% 61% 56% 57% 61% 55% 57% 60% 56% Specificity 70% 71% 53% 71% 71% 53% 71% 71% 54% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 32% 35% 33% 32% 35% 32% 31% 35% 33% Specificity 95% 94% 80% 94% 94% 80% 94% 94% 81% OR Quartile 2 2.36 2.95 1.18 2.62 2.95 1.46 2.25 2.56 1.61 p Value 0.054 0.012 0.71 0.032 0.012 0.40 0.076 0.028 0.29 Lower limit of 95% CI 0.987 1.27 0.491 1.08 1.27 0.606 0.919 1.10 0.671 Upper limit of 95% CI 5.66 6.85 2.86 6.31 6.85 3.51 5.48 5.93 3.87 OR Quartile 3 3.15 3.91 1.42 3.32 3.91 1.40 3.12 3.70 1.47 p Value 0.0048 3.0E−4 0.31 0.0042 3.0E−4 0.32 0.0068 5.2E−4 0.25 Lower limit of 95% CI 1.42 1.87 0.725 1.46 1.87 0.725 1.37 1.77 0.766 Upper limit of 95% CI 7.00 8.18 2.78 7.54 8.18 2.71 7.12 7.76 2.84 OR Quartile 4 8.29 8.26 1.97 7.62 8.26 1.80 7.29 7.95 2.16 p Value 0.0051 8.3E−4 0.078 0.0072 8.3E−4 0.12 0.0086 0.0010 0.046 Lower limit of 95% CI 1.89 2.39 0.925 1.73 2.39 0.850 1.66 2.31 1.01 Upper limit of 95% CI 36.4 28.5 4.21 33.5 28.5 3.82 32.1 27.4 4.59

TABLE 11.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000473 0.866 0.000473 0.862 0.000473 0.862 Average 2.71 10.2 2.81 9.97 2.81 9.97 Stdev 15.4 20.1 15.8 20.0 15.8 20.0 p (t-test) 0.022 0.030 0.030 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 51 98 49 100 49 100 sCr only Median 0.000473 0.927 0.000473 0.899 0.000473 0.899 Average 2.66 10.8 2.71 10.7 2.71 10.7 Stdev 14.8 20.7 14.9 20.6 14.9 20.6 p (t-test) 0.011 0.013 0.013 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 56 91 55 92 55 92 UO only Median 0.122 0.869 0.118 0.866 0.118 0.866 Average 7.03 8.82 6.02 10.3 6.02 10.3 Stdev 20.7 15.9 17.4 21.5 17.4 21.5 p (t-test) 0.59 0.19 0.19 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 59.1 110 120 110 120 n (Patient) 94 50 89 55 89 55 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.74 0.59 0.70 0.73 0.60 0.70 0.73 0.60 SE 0.042 0.040 0.051 0.043 0.041 0.049 0.043 0.041 0.049 p Value 9.2E−7 3.9E−9 0.082 3.1E−6 3.3E−8 0.045 3.1E−6 3.3E−8 0.045 nCohort Recovered 51 56 94 49 55 89 49 55 89 nCohort Non-recovered 98 91 50 100 92 55 100 92 55 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 85% 87% 84% 84% 86% 85% 84% 86% 85% Specificity 25% 27% 20% 24% 25% 21% 24% 25% 21% Cutoff Quartile 3 0.170 0.170 0.218 0.170 0.170 0.218 0.170 0.170 0.218 Sensitivity 60% 64% 56% 60% 63% 56% 60% 63% 56% Specificity 69% 71% 53% 69% 71% 54% 69% 71% 54% Cutoff Quartile 4 2.99 3.02 3.01 2.99 3.02 3.01 2.99 3.02 3.01 Sensitivity 37% 37% 36% 36% 37% 36% 36% 37% 36% Specificity 96% 95% 81% 96% 95% 82% 96% 95% 82% OR Quartile 2 1.89 2.41 1.33 1.70 2.08 1.59 1.70 2.08 1.59 p Value 0.13 0.042 0.54 0.22 0.090 0.31 0.22 0.090 0.31 Lower limit of 95% CI 0.820 1.03 0.536 0.733 0.892 0.645 0.733 0.892 0.645 Upper limit of 95% CI 4.37 5.62 3.30 3.95 4.83 3.94 3.95 4.83 3.94 OR Quartile 3 3.31 4.39 1.45 3.40 4.16 1.51 3.40 4.16 1.51 p Value 0.0011 5.6E−5 0.29 9.8E−4 1.0E−4 0.23 9.8E−4 1.0E−4 0.23 Lower limit of 95% CI 1.62 2.14 0.725 1.64 2.02 0.769 1.64 2.02 0.769 Upper limit of 95% CI 6.78 9.03 2.88 7.04 8.54 2.97 7.04 8.54 2.97 OR Quartile 4 14.2 10.5 2.38 13.2 10.2 2.61 13.2 10.2 2.61 p Value 4.1E−4 1.9E−4 0.028 5.9E−4 2.4E−4 0.015 5.9E−4 2.4E−4 0.015 Lower limit of 95% CI 3.26 3.05 1.10 3.03 2.95 1.21 3.03 2.95 1.21 Upper limit of 95% CI 62.0 36.4 5.14 57.7 35.1 5.64 57.7 35.1 5.64

TABLE 11.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0328 0.866 0.0328 0.866 0.0328 0.866 Average 3.63 10.9 3.63 10.9 3.52 10.8 Stdev 14.7 21.4 14.7 21.4 14.9 21.2 p (t-test) 0.023 0.023 0.023 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 63 83 63 83 61 85 sCr only Median 0.0328 0.899 0.0328 0.899 0.0169 0.927 Average 3.61 11.4 3.61 11.4 3.46 11.4 Stdev 14.3 21.9 14.3 21.9 14.4 21.8 p (t-test) 0.014 0.014 0.012 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 67 78 67 78 66 79 UO only Median 0.146 0.899 0.146 0.899 0.165 0.866 Average 7.31 10.6 7.31 10.6 7.58 10.0 Stdev 19.2 21.6 19.2 21.6 19.5 21.1 p (t-test) 0.37 0.37 0.51 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 80 48 80 48 77 51 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.65 0.67 0.60 0.65 0.67 0.60 0.67 0.68 0.59 SE 0.045 0.044 0.052 0.045 0.044 0.052 0.044 0.044 0.052 p Value 5.9E−4 1.4E−4 0.051 5.9E−4 1.4E−4 0.051 1.6E−4 4.3E−5 0.078 nCohort Recovered 63 67 80 63 67 80 61 66 77 nCohort Non-recovered 83 78 48 83 78 48 85 79 51 Cutoff Quartile 2 0.000339 0.000339 0.000471 0.000339 0.000339 0.000471 0.000339 0.000339 0.000471 Sensitivity 83% 85% 81% 83% 85% 81% 84% 85% 82% Specificity 21% 22% 28% 21% 22% 28% 21% 23% 29% Cutoff Quartile 3 0.168 0.165 0.399 0.168 0.165 0.399 0.168 0.165 0.399 Sensitivity 59% 62% 58% 59% 62% 58% 60% 62% 57% Specificity 62% 61% 55% 62% 61% 55% 64% 62% 55% Cutoff Quartile 4 3.04 3.05 4.89 3.04 3.05 4.89 3.04 3.05 4.89 Sensitivity 35% 36% 33% 35% 36% 33% 35% 37% 31% Specificity 87% 87% 80% 87% 87% 80% 89% 88% 79% OR Quartile 2 1.28 1.59 1.64 1.28 1.59 1.64 1.37 1.64 1.87 p Value 0.56 0.28 0.27 0.56 0.28 0.27 0.46 0.25 0.16 Lower limit of 95% CI 0.554 0.684 0.685 0.554 0.684 0.685 0.594 0.708 0.779 Upper limit of 95% CI 2.96 3.68 3.94 2.96 3.68 3.94 3.18 3.81 4.47 OR Quartile 3 2.34 2.52 1.71 2.34 2.52 1.71 2.66 2.68 1.58 p Value 0.013 0.0068 0.15 0.013 0.0068 0.15 0.0048 0.0041 0.21 Lower limit of 95% CI 1.20 1.29 0.830 1.20 1.29 0.830 1.35 1.37 0.775 Upper limit of 95% CI 4.58 4.93 3.53 4.58 4.93 3.53 5.24 5.25 3.23 OR Quartile 4 3.69 3.61 2.00 3.69 3.61 2.00 4.21 4.20 1.74 p Value 0.0032 0.0028 0.095 0.0032 0.0028 0.095 0.0018 0.0012 0.18 Lower limit of 95% CI 1.55 1.56 0.887 1.55 1.56 0.887 1.70 1.76 0.777 Upper limit of 95% CI 8.80 8.37 4.51 8.80 8.37 4.51 10.4 10.0 3.91

TABLE 11.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000473 1.21 0.000473 1.14 0.000473 1.13 Average 3.27 12.6 3.26 12.2 3.21 11.9 Stdev 13.9 22.7 14.3 22.2 14.5 21.8 p (t-test) 0.0035 0.0057 0.0075 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 71 71 67 75 64 78 sCr only Median 0.00102 1.21 0.000747 1.28 0.000473 1.35 Average 3.36 12.8 3.22 12.8 3.09 12.8 Stdev 13.8 23.0 13.8 22.8 13.9 22.6 p (t-test) 0.0032 0.0028 0.0024 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 73 69 72 70 71 71 UO only Median 0.0873 1.13 0.0873 0.872 0.118 0.866 Average 9.20 8.52 8.23 9.89 8.55 9.52 Stdev 23.8 14.7 20.6 21.1 20.7 20.8 p (t-test) 0.86 0.67 0.80 Min 0.000212 0.000286 0.000212 0.000286 0.000212 0.000286 Max 120 50.9 110 120 110 120 n (Patient) 74 45 68 51 67 53 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.70 0.63 0.72 0.71 0.61 0.72 0.72 0.59 SE 0.043 0.044 0.054 0.043 0.043 0.053 0.042 0.043 0.053 p Value 6.7E−7 3.5E−6 0.019 4.2E−7 8.0E−7 0.038 3.6E−7 1.6E−7 0.097 nCohort Recovered 71 73 74 67 72 68 64 71 67 nCohort Non-recovered 71 69 45 75 70 51 78 71 53 Cutoff Quartile 2 0.000365 0.000365 0.000471 0.000365 0.000365 0.000471 0.000365 0.000365 0.000471 Sensitivity 82% 81% 84% 83% 81% 84% 82% 82% 83% Specificity 32% 32% 28% 34% 32% 29% 34% 32% 28% Cutoff Quartile 3 0.205 0.205 0.404 0.205 0.205 0.404 0.205 0.205 0.424 Sensitivity 66% 65% 62% 65% 66% 59% 64% 66% 57% Specificity 66% 64% 57% 67% 65% 56% 67% 66% 55% Cutoff Quartile 4 3.58 3.58 4.63 3.58 3.58 4.63 3.58 3.58 4.89 Sensitivity 39% 39% 33% 39% 40% 31% 38% 41% 28% Specificity 89% 88% 80% 90% 89% 79% 91% 90% 78% OR Quartile 2 2.14 1.98 2.15 2.49 2.06 2.24 2.39 2.14 1.94 p Value 0.056 0.086 0.11 0.022 0.070 0.085 0.027 0.056 0.15 Lower limit of 95% CI 0.980 0.909 0.831 1.14 0.943 0.895 1.10 0.980 0.793 Upper limit of 95% CI 4.66 4.32 5.57 5.45 4.49 5.61 5.20 4.66 4.72 OR Quartile 3 3.84 3.39 2.16 3.85 3.60 1.81 3.66 3.84 1.61 p Value 1.5E−4 5.2E−4 0.046 1.5E−4 2.8E−4 0.11 2.7E−4 1.5E−4 0.20 Lower limit of 95% CI 1.91 1.70 1.01 1.92 1.80 0.868 1.82 1.91 0.778 Upper limit of 95% CI 7.69 6.75 4.61 7.74 7.20 3.77 7.34 7.69 3.32 OR Quartile 4 5.13 4.57 1.97 5.40 5.33 1.76 6.04 6.31 1.37 p Value 2.6E−4 4.5E−4 0.11 2.8E−4 1.8E−4 0.18 2.3E−4 7.6E−5 0.46 Lower limit of 95% CI 2.14 1.96 0.849 2.17 2.22 0.766 2.32 2.53 0.597 Upper limit of 95% CI 12.3 10.7 4.55 13.4 12.8 4.06 15.7 15.7 3.13

TABLE 11.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0328 1.13 0.0328 1.13 0.0328 1.03 Average 6.41 9.62 6.48 9.45 6.55 9.29 Stdev 20.4 16.2 20.5 16.1 20.6 16.0 p (t-test) 0.32 0.35 0.39 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 59.1 120 59.1 120 59.1 n (Patient) 93 56 92 57 91 58 sCr only Median 0.00102 1.28 0.00102 1.28 0.00102 1.28 Average 3.04 13.4 3.04 13.4 3.04 13.4 Stdev 12.9 23.4 12.9 23.4 12.9 23.4 p (t-test) 8.0E−4 8.0E−4 8.0E−4 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 83 66 83 66 83 66 UO only Median 0.0851 1.13 0.0851 1.13 0.0873 1.03 Average 6.94 9.19 6.94 9.19 7.08 8.82 Stdev 20.6 15.3 20.6 15.3 20.8 15.1 p (t-test) 0.50 0.50 0.60 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 50.9 120 50.9 120 50.9 n (Patient) 100 48 100 48 98 50 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.72 0.64 0.67 0.72 0.64 0.66 0.72 0.63 SE 0.047 0.043 0.050 0.047 0.043 0.050 0.047 0.043 0.050 p Value 2.2E−4 4.0E−7 0.0051 3.9E−4 4.0E−7 0.0051 5.9E−4 4.0E−7 0.011 nCohort Recovered 93 83 100 92 83 100 91 83 98 nCohort Non-recovered 56 66 48 57 66 48 58 66 50 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 89% 88% 88% 89% 88% 88% 90% 88% 88% Specificity 24% 24% 21% 24% 24% 21% 24% 24% 21% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 64% 65% 62% 63% 65% 62% 62% 65% 60% Specificity 58% 61% 56% 58% 61% 56% 57% 61% 55% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 36% 41% 38% 35% 41% 38% 34% 41% 36% Specificity 81% 87% 81% 80% 87% 81% 80% 87% 81% OR Quartile 2 2.58 2.30 1.86 2.67 2.30 1.86 2.76 2.30 2.00 p Value 0.056 0.068 0.21 0.048 0.068 0.21 0.040 0.068 0.17 Lower limit of 95% CI 0.976 0.941 0.697 1.01 0.941 0.697 1.05 0.941 0.751 Upper limit of 95% CI 6.83 5.63 4.97 7.06 5.63 4.97 7.30 5.63 5.33 OR Quartile 3 2.49 2.98 2.12 2.33 2.98 2.12 2.18 2.98 1.84 p Value 0.0089 0.0015 0.037 0.015 0.0015 0.037 0.023 0.0015 0.084 Lower limit of 95% CI 1.26 1.52 1.05 1.18 1.52 1.05 1.11 1.52 0.922 Upper limit of 95% CI 4.94 5.84 4.29 4.59 5.84 4.29 4.28 5.84 3.68 OR Quartile 4 2.31 4.53 2.56 2.22 4.53 2.56 2.13 4.53 2.34 p Value 0.028 2.2E−4 0.017 0.037 2.2E−4 0.017 0.047 2.2E−4 0.029 Lower limit of 95% CI 1.09 2.03 1.19 1.05 2.03 1.19 1.01 2.03 1.09 Upper limit of 95% CI 4.90 10.1 5.52 4.70 10.1 5.52 4.51 10.1 5.02

Example 12. Use of Insulin-Like Growth Factor-Binding Protein 6 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 6 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is risk of injury (R), injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 12.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 7.89 86.4 7.31 85.4 7.31 85.4 Average 70.0 296 71.2 294 71.2 294 Stdev 142 551 150 549 150 549 p (t-test) 0.20 0.23 0.23 Min 0.352 0.259 0.352 0.259 0.352 0.259 Max 460 3600 460 3600 460 3600 n (Patient) 10 139 9 140 9 140 sCr only Median 8.47 93.9 8.47 93.8 8.47 93.8 Average 70.6 319 72.8 317 72.8 317 Stdev 141 572 144 570 144 570 p (t-test) 0.041 0.049 0.049 Min 0.352 0.259 0.352 0.259 0.352 0.259 Max 532 3600 532 3600 532 3600 n (Patient) 23 126 22 127 22 127 UO only Median 63.7 98.2 63.7 93.8 61.7 94.8 Average 212 354 213 340 181 360 Stdev 381 655 381 635 320 648 p (t-test) 0.11 0.16 0.045 Min 0.259 1.14 0.259 1.14 0.259 1.14 Max 1830 3600 1830 3600 1650 3600 n (Patient) 75 73 67 81 64 84 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.74 0.58 0.75 0.74 0.58 0.75 0.74 0.59 SE 0.069 0.049 0.047 0.069 0.050 0.047 0.069 0.050 0.047 p Value 6.7E−4 8.4E−7 0.073 2.7E−4 1.6E−6 0.081 2.7E−4 1.6E−6 0.042 nCohort Recovered 10 23 75 9 22 67 9 22 64 nCohort Non-recovered 139 126 73 140 127 81 140 127 84 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 78% 81% 79% 78% 81% 81% 78% 81% 81% Specificity 60% 57% 29% 67% 59% 33% 67% 59% 33% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 53% 56% 56% 52% 55% 53% 52% 55% 54% Specificity 80% 78% 56% 78% 77% 54% 78% 77% 55% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 27% 29% 30% 26% 28% 28% 26% 28% 30% Specificity 90% 91% 80% 89% 91% 79% 89% 91% 81% OR Quartile 2 5.23 5.52 1.61 7.03 6.20 2.15 7.03 6.20 2.08 p Value 0.015 3.5E−4 0.22 0.0080 1.9E−4 0.048 0.0080 1.9E−4 0.058 Lower limit of 95% CI 1.39 2.17 0.755 1.66 2.38 1.01 1.66 2.38 0.976 Upper limit of 95% CI 19.7 14.1 3.41 29.7 16.2 4.59 29.7 16.2 4.41 OR Quartile 3 4.42 4.50 1.63 3.81 4.18 1.31 3.81 4.18 1.39 p Value 0.066 0.0050 0.14 0.10 0.0080 0.41 0.10 0.0080 0.32 Lower limit of 95% CI 0.907 1.57 0.852 0.765 1.45 0.687 0.765 1.45 0.725 Upper limit of 95% CI 21.6 12.9 3.12 19.0 12.0 2.51 19.0 12.0 2.67 OR Quartile 4 3.26 4.20 1.73 2.87 3.96 1.50 2.87 3.96 1.84 p Value 0.27 0.061 0.16 0.33 0.073 0.30 0.33 0.073 0.13 Lower limit of 95% CI 0.400 0.936 0.811 0.348 0.879 0.701 0.348 0.879 0.839 Upper limit of 95% CI 26.7 18.8 3.67 23.8 17.8 3.22 23.8 17.8 4.02

TABLE 12.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 15.1 95.7 12.9 93.8 8.47 93.6 Average 52.2 329 55.2 320 57.0 317 Stdev 94.7 577 102 570 104 569 p (t-test) 0.016 0.032 0.039 Min 0.352 0.259 0.352 0.259 0.352 0.259 Max 460 3600 460 3600 460 3600 n (Patient) 26 123 22 127 21 128 sCr only Median 19.4 98.7 17.2 97.0 16.2 95.7 Average 72.5 350 74.1 345 75.9 342 Stdev 124 599 128 595 129 593 p (t-test) 0.0073 0.011 0.013 Min 0.352 0.259 0.352 0.259 0.352 0.259 Max 532 3600 532 3600 532 3600 n (Patient) 35 112 33 114 32 115 UO only Median 59.7 99.2 59.3 98.7 58.8 99.2 Average 213 380 188 384 177 386 Stdev 372 697 320 688 315 677 p (t-test) 0.067 0.030 0.021 Min 0.259 2.24 0.259 2.24 0.259 2.24 Max 1830 3600 1650 3600 1650 3600 n (Patient) 81 63 72 72 69 75 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.73 0.60 0.74 0.73 0.61 0.74 0.72 0.62 SE 0.046 0.045 0.048 0.050 0.045 0.047 0.051 0.046 0.046 p Value 8.4E−8 4.5E−7 0.037 1.2E−6 6.6E−7 0.023 3.3E−6 1.8E−6 0.0076 nCohort Recovered 26 35 81 22 33 72 21 32 69 nCohort Non-recovered 123 112 63 127 114 72 128 115 75 Cutoff Quartile 2 17.2 17.5 16.5 17.2 17.5 16.5 17.2 17.5 16.5 Sensitivity 80% 82% 83% 80% 82% 82% 80% 82% 83% Specificity 50% 49% 31% 50% 52% 32% 52% 50% 33% Cutoff Quartile 3 78.3 83.5 80.9 78.3 83.5 80.9 78.3 83.5 80.9 Sensitivity 57% 58% 57% 55% 57% 56% 55% 57% 56% Specificity 81% 74% 56% 77% 73% 56% 76% 72% 57% Cutoff Quartile 4 331 332 331 331 332 331 331 332 331 Sensitivity 30% 31% 30% 29% 31% 29% 29% 30% 31% Specificity 96% 94% 79% 95% 94% 79% 95% 94% 81% OR Quartile 2 4.12 4.34 2.11 3.88 4.99 2.13 4.32 4.48 2.38 p Value 0.0018 4.5E−4 0.068 0.0047 1.6E−4 0.057 0.0028 4.6E−4 0.029 Lower limit of 95% CI 1.70 1.91 0.945 1.52 2.16 0.978 1.65 1.93 1.09 Upper limit of 95% CI 10.0 9.87 4.71 9.95 11.5 4.64 11.3 10.4 5.20 OR Quartile 3 5.55 4.00 1.67 4.18 3.54 1.56 3.86 3.32 1.65 p Value 0.0012 0.0013 0.13 0.0080 0.0036 0.18 0.013 0.0059 0.13 Lower limit of 95% CI 1.96 1.71 0.858 1.45 1.51 0.810 1.33 1.41 0.856 Upper limit of 95% CI 15.7 9.31 3.24 12.0 8.29 3.02 11.2 7.81 3.20 OR Quartile 4 10.8 7.50 1.63 8.63 6.87 1.56 8.13 6.56 1.91 p Value 0.022 0.0077 0.21 0.039 0.011 0.25 0.045 0.013 0.10 Lower limit of 95% CI 1.40 1.70 0.761 1.12 1.56 0.730 1.05 1.49 0.875 Upper limit of 95% CI 82.4 33.0 3.47 66.5 30.3 3.35 62.8 29.0 4.15

TABLE 12.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 20.6 103 20.8 101 26.8 98.2 Average 54.4 360 47.7 354 51.6 345 Stdev 80.0 605 62.2 599 63.8 593 p (t-test) 0.0027 0.0035 0.0069 Min 0.352 0.259 0.352 0.259 1.13 0.259 Max 367 3600 225 3600 225 3600 n (Patient) 37 109 34 112 31 115 sCr only Median 21.0 106 21.0 106 21.0 106 Average 70.6 380 64.9 377 64.9 377 Stdev 110 626 102 621 102 621 p (t-test) 0.0013 0.0014 0.0014 Min 0.352 0.259 0.352 0.259 0.352 0.259 Max 532 3600 532 3600 532 3600 n (Patient) 45 100 43 102 43 102 UO only Median 61.7 114 61.7 114 63.7 99.2 Average 230 410 224 404 246 368 Stdev 404 713 406 695 424 667 p (t-test) 0.076 0.074 0.23 Min 0.259 2.24 0.259 2.24 0.259 0.352 Max 2160 3600 2160 3600 2160 3600 n (Patient) 70 58 66 62 59 69 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.73 0.62 0.75 0.74 0.62 0.72 0.74 0.58 SE 0.042 0.042 0.050 0.043 0.042 0.050 0.046 0.042 0.050 p Value 2.9E−9 2.7E−8 0.020 3.7E−9 9.2E−9 0.014 1.8E−6 9.2E−9 0.13 nCohort Recovered 37 45 70 34 43 66 31 43 59 nCohort Non-recovered 109 100 58 112 102 62 115 102 69 Cutoff Quartile 2 17.4 17.2 20.7 17.4 17.2 20.7 17.4 17.2 20.7 Sensitivity 82% 83% 83% 80% 83% 82% 79% 83% 80% Specificity 46% 42% 31% 44% 44% 32% 42% 44% 31% Cutoff Quartile 3 80.9 83.5 87.1 80.9 83.5 87.1 80.9 83.5 87.1 Sensitivity 59% 60% 57% 58% 60% 56% 57% 60% 54% Specificity 76% 71% 56% 76% 72% 56% 74% 72% 54% Cutoff Quartile 4 325 331 369 325 331 369 325 331 369 Sensitivity 33% 35% 29% 33% 35% 29% 32% 35% 26% Specificity 97% 96% 79% 100%  98% 79% 100%  98% 76% OR Quartile 2 3.78 3.57 2.20 3.23 3.96 2.16 2.74 3.96 1.72 p Value 0.0013 0.0016 0.068 0.0052 7.0E−4 0.069 0.019 7.0E−4 0.19 Lower limit of 95% CI 1.69 1.62 0.942 1.42 1.79 0.941 1.18 1.79 0.770 Upper limit of 95% CI 8.49 7.85 5.14 7.35 8.77 4.97 6.36 8.77 3.87 OR Quartile 3 4.42 3.69 1.66 4.49 3.84 1.65 3.74 3.84 1.37 p Value 5.4E−4 7.4E−4 0.16 7.8E−4 6.6E−4 0.16 0.0035 6.6E−4 0.38 Lower limit of 95% CI 1.91 1.73 0.823 1.87 1.77 0.822 1.54 1.77 0.682 Upper limit of 95% CI 10.3 7.88 3.35 10.8 8.34 3.33 9.06 8.34 2.75 OR Quartile 4 17.8 11.6 1.52 34.3 22.9 1.52 30.1 22.9 1.13 p Value 0.0054 0.0011 0.31 0.014 0.0024 0.31 0.018 0.0024 0.76 Lower limit of 95% CI 2.34 2.65 0.681 2.04 3.03 0.679 1.79 3.03 0.507 Upper limit of 95% CI 135 50.7 3.40 575 173 3.40 505 173 2.54

TABLE 12.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 22.2 108 24.5 98.7 24.5 98.7 Average 100 370 109 354 112 348 Stdev 239 619 253 608 259 604 p (t-test) 0.0065 0.018 0.024 Min 0.352 0.259 1.13 0.259 1.13 0.259 Max 1440 3600 1440 3600 1440 3600 n (Patient) 43 99 38 104 36 106 sCr only Median 33.4 106 33.4 106 33.4 106 Average 111 385 111 385 113 378 Stdev 232 638 232 638 236 633 p (t-test) 0.0040 0.0040 0.0059 Min 0.352 0.259 0.352 0.259 0.352 0.259 Max 1440 3600 1440 3600 1440 3600 n (Patient) 50 92 50 92 48 94 UO only Median 61.3 117 71.0 99.2 86.1 94.8 Average 245 403 266 365 289 348 Stdev 433 712 456 676 470 656 p (t-test) 0.14 0.36 0.59 Min 0.259 2.91 0.259 0.352 0.259 0.352 Max 2160 3600 2160 3600 2160 3600 n (Patient) 62 57 54 65 50 70 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.70 0.63 0.70 0.70 0.58 0.70 0.69 0.55 SE 0.043 0.044 0.051 0.046 0.044 0.052 0.047 0.045 0.053 p Value 1.4E−7 7.0E−6 0.011 1.0E−5 7.0E−6 0.11 2.3E−5 1.7E−5 0.38 nCohort Recovered 43 50 62 38 50 54 36 48 50 nCohort Non-recovered 99 92 57 104 92 65 106 94 70 Cutoff Quartile 2 17.4 17.4 25.6 17.4 17.4 25.6 17.4 17.4 25.8 Sensitivity 82% 82% 84% 81% 82% 80% 80% 81% 79% Specificity 42% 38% 34% 42% 38% 31% 42% 38% 30% Cutoff Quartile 3 80.9 80.9 87.9 80.9 80.9 87.9 80.9 80.9 90.6 Sensitivity 60% 60% 58% 58% 60% 54% 58% 60% 51% Specificity 72% 68% 56% 71% 68% 54% 72% 69% 52% Cutoff Quartile 4 332 332 364 332 332 364 332 332 369 Sensitivity 33% 35% 28% 32% 35% 26% 31% 34% 24% Specificity 93% 92% 77% 92% 92% 76% 92% 92% 74% OR Quartile 2 3.24 2.70 2.73 3.05 2.70 1.84 2.89 2.53 1.57 p Value 0.0036 0.012 0.026 0.0067 0.012 0.15 0.011 0.019 0.29 Lower limit of 95% CI 1.47 1.24 1.13 1.36 1.24 0.796 1.28 1.16 0.684 Upper limit of 95% CI 7.16 5.88 6.62 6.85 5.88 4.24 6.54 5.52 3.61 OR Quartile 3 3.81 3.16 1.78 3.35 3.16 1.35 3.52 3.24 1.15 p Value 7.5E−4 0.0019 0.12 0.0031 0.0019 0.41 0.0028 0.0017 0.71 Lower limit of 95% CI 1.75 1.53 0.862 1.50 1.53 0.656 1.54 1.55 0.555 Upper limit of 95% CI 8.30 6.53 3.69 7.46 6.53 2.79 8.04 6.77 2.37 OR Quartile 4 6.67 6.13 1.34 5.42 6.13 1.12 4.97 5.68 0.913 p Value 0.0028 0.0013 0.49 0.0080 0.0013 0.79 0.012 0.0021 0.83 Lower limit of 95% CI 1.92 2.03 0.584 1.55 2.03 0.485 1.42 1.87 0.396 Upper limit of 95% CI 23.2 18.6 3.07 18.9 18.6 2.57 17.4 17.2 2.10

TABLE 12.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 44.2 117 40.8 118 40.8 118 Average 206 382 207 379 207 379 Stdev 393 675 396 670 396 670 p (t-test) 0.047 0.052 0.052 Min 0.259 2.91 0.259 2.91 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 86 63 85 64 85 64 sCr only Median 36.8 123 36.8 119 36.8 119 Average 137 412 140 402 140 402 Stdev 322 649 326 644 326 644 p (t-test) 0.0015 0.0026 0.0026 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 71 78 69 80 69 80 UO only Median 58.9 113 58.9 113 58.9 113 Average 214 408 217 396 216 391 Stdev 380 733 383 722 386 711 p (t-test) 0.035 0.051 0.054 Min 0.259 3.02 0.259 2.91 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 96 52 94 54 92 56 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.65 0.72 0.62 0.66 0.71 0.61 0.66 0.71 0.61 SE 0.046 0.041 0.049 0.046 0.042 0.049 0.046 0.042 0.049 p Value 8.2E−4 8.6E−8 0.019 5.5E−4 4.6E−7 0.031 5.5E−4 4.6E−7 0.028 nCohort Recovered 86 71 96 85 69 94 85 69 92 nCohort Non-recovered 63 78 52 64 80 54 64 80 56 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 84% 86% 83% 84% 85% 81% 84% 85% 80% Specificity 31% 37% 29% 32% 36% 29% 32% 36% 28% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 65% 67% 62% 66% 65% 61% 66% 65% 61% Specificity 60% 68% 56% 61% 67% 56% 61% 67% 57% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 33% 38% 31% 33% 38% 30% 33% 38% 30% Specificity 80% 89% 78% 80% 88% 78% 80% 88% 78% OR Quartile 2 2.43 3.52 1.97 2.51 3.22 1.77 2.51 3.22 1.61 p Value 0.033 0.0020 0.12 0.027 0.0035 0.17 0.027 0.0035 0.24 Lower limit of 95% CI 1.07 1.58 0.847 1.11 1.47 0.782 1.11 1.47 0.724 Upper limit of 95% CI 5.48 7.83 4.57 5.68 7.07 4.02 5.68 7.07 3.59 OR Quartile 3 2.85 4.17 2.06 3.01 3.71 2.03 3.01 3.71 2.01 p Value 0.0023 4.3E−5 0.040 0.0014 1.5E−4 0.042 0.0014 1.5E−4 0.043 Lower limit of 95% CI 1.45 2.10 1.03 1.53 1.88 1.03 1.53 1.88 1.02 Upper limit of 95% CI 5.60 8.28 4.10 5.91 7.33 4.02 5.91 7.33 3.95 OR Quartile 4 2.03 4.92 1.59 1.95 4.58 1.46 1.95 4.58 1.57 p Value 0.063 3.1E−4 0.23 0.078 5.7E−4 0.33 0.078 5.7E−4 0.24 Lower limit of 95% CI 0.963 2.07 0.741 0.928 1.93 0.685 0.928 1.93 0.738 Upper limit of 95% CI 4.28 11.7 3.40 4.11 10.9 3.13 4.11 10.9 3.34

Example 13. Use of Insulin-Like Growth Factor-Binding Protein 6 for Evaluating Renal Status in Patients Admitted to the ICU: Recovery to RIFLE 0 and R from RIFLE I and F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 6 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output during a period starting at 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection. Two cohorts are defined to represent a “recovered” and a “non-recovered” population. “Recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R). “Non-recovered” indicates those patients whose maximum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F). If a patient dies or is placed on renal replacement therapy (RRT) at any time from sample collection to 12, 24, 48, or 72 hours after sample collection or at any time within 7 days after sample collection, the patient is considered “non-recovered”.

The ability to distinguish the “recovered” and “non-recovered” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 13.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 12 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 20.6 98.7 17.2 97.0 18.9 95.7 Average 125 332 131 327 135 324 Stdev 368 573 378 569 383 568 p (t-test) 0.042 0.059 0.070 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 37 112 35 114 34 115 sCr only Median 21.0 106 21.0 106 21.6 103 Average 121 359 121 359 124 355 Stdev 328 599 328 599 331 597 p (t-test) 0.011 0.011 0.013 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 49 100 49 100 48 101 UO only Median 58.5 121 58.9 104 58.8 108 Average 212 405 223 369 206 386 Stdev 405 699 416 672 384 682 p (t-test) 0.034 0.11 0.043 Min 0.259 2.91 0.259 2.91 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 94 54 88 60 85 63 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.73 0.72 0.65 0.72 0.72 0.62 0.71 0.72 0.63 SE 0.044 0.042 0.048 0.045 0.042 0.048 0.046 0.042 0.047 p Value 1.9E−7 6.5E−8 0.0022 5.0E−7 6.5E−8 0.015 2.8E−6 3.6E−7 0.0065 nCohort Recovered 37 49 94 35 49 88 34 48 85 nCohort Non-recovered 112 100 54 114 100 60 115 101 63 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 82% 84% 85% 82% 84% 85% 82% 83% 86% Specificity 46% 43% 31% 49% 43% 32% 47% 42% 33% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 59% 61% 65% 58% 61% 60% 57% 60% 60% Specificity 76% 71% 59% 74% 71% 57% 74% 71% 58% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 31% 34% 35% 31% 34% 32% 30% 34% 32% Specificity 92% 92% 81% 91% 92% 80% 91% 92% 80% OR Quartile 2 3.91 3.94 2.57 4.44 3.94 2.64 3.98 3.53 2.95 p Value 9.3E−4 5.6E−4 0.034 3.7E−4 5.6E−4 0.023 0.0010 0.0014 0.011 Lower limit of 95% CI 1.74 1.81 1.08 1.96 1.81 1.14 1.75 1.63 1.27 Upper limit of 95% CI 8.77 8.58 6.12 10.1 8.58 6.12 9.06 7.66 6.81 OR Quartile 3 4.46 3.91 2.60 3.97 3.91 1.97 3.74 3.70 2.07 p Value 4.8E−4 3.0E−4 0.0069 0.0014 3.0E−4 0.046 0.0023 5.2E−4 0.032 Lower limit of 95% CI 1.93 1.87 1.30 1.71 1.87 1.01 1.60 1.77 1.07 Upper limit of 95% CI 10.3 8.18 5.20 9.24 8.18 3.84 8.73 7.76 4.02 OR Quartile 4 5.15 5.80 2.29 4.73 5.80 1.80 4.52 5.58 1.86 p Value 0.0099 0.0018 0.032 0.015 0.0018 0.12 0.018 0.0023 0.11 Lower limit of 95% CI 1.48 1.92 1.07 1.36 1.92 0.850 1.30 1.85 0.878 Upper limit of 95% CI 17.9 17.5 4.90 16.5 17.5 3.82 15.8 16.8 3.94

TABLE 13.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 26.8 110 26.8 109 26.8 109 Average 121 363 125 357 125 357 Stdev 323 603 329 599 329 599 p (t-test) 0.0084 0.013 0.013 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 51 98 49 100 49 100 sCr only Median 28.9 111 30.9 110 30.9 110 Average 125 382 127 378 127 378 Stdev 314 621 316 618 316 618 p (t-test) 0.0047 0.0060 0.0060 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 56 91 55 92 55 92 UO only Median 58.5 114 58.8 109 58.8 109 Average 217 414 204 417 204 417 Stdev 405 724 377 722 377 722 p (t-test) 0.038 0.022 0.022 Min 0.259 2.91 0.259 2.91 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 94 50 89 55 89 55 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.72 0.64 0.71 0.71 0.64 0.71 0.71 0.64 SE 0.042 0.042 0.050 0.043 0.042 0.048 0.043 0.042 0.048 p Value 1.7E−7 2.4E−7 0.0040 1.2E−6 1.2E−6 0.0036 1.2E−6 1.2E−6 0.0036 nCohort Recovered 51 56 94 49 55 89 49 55 89 nCohort Non-recovered 98 91 50 100 92 55 100 92 55 Cutoff Quartile 2 17.2 17.5 16.5 17.2 17.5 16.5 17.2 17.5 16.5 Sensitivity 84% 85% 86% 83% 84% 87% 83% 84% 87% Specificity 41% 41% 31% 41% 40% 33% 41% 40% 33% Cutoff Quartile 3 78.3 83.5 80.9 78.3 83.5 80.9 78.3 83.5 80.9 Sensitivity 62% 63% 64% 61% 62% 62% 61% 62% 62% Specificity 73% 70% 57% 71% 69% 57% 71% 69% 57% Cutoff Quartile 4 331 332 331 331 332 331 331 332 331 Sensitivity 35% 35% 34% 34% 35% 33% 34% 35% 33% Specificity 92% 91% 80% 92% 91% 80% 92% 91% 80% OR Quartile 2 3.59 3.83 2.74 3.37 3.42 3.31 3.37 3.42 3.31 p Value 0.0012 7.3E−4 0.030 0.0021 0.0018 0.0097 0.0021 0.0018 0.0097 Lower limit of 95% CI 1.66 1.76 1.10 1.56 1.58 1.34 1.56 1.58 1.34 Upper limit of 95% CI 7.77 8.36 6.81 7.29 7.41 8.22 7.29 7.41 8.22 OR Quartile 3 4.36 3.85 2.40 3.91 3.64 2.17 3.91 3.64 2.17 p Value 9.3E−5 2.0E−4 0.015 3.0E−4 3.6E−4 0.027 3.0E−4 3.6E−4 0.027 Lower limit of 95% CI 2.08 1.89 1.18 1.87 1.79 1.09 1.87 1.79 1.09 Upper limit of 95% CI 9.12 7.83 4.87 8.18 7.41 4.32 8.18 7.41 4.32 OR Quartile 4 6.24 5.53 2.03 5.80 5.33 1.92 5.80 5.33 1.92 p Value 0.0011 9.5E−4 0.072 0.0018 0.0012 0.095 0.0018 0.0012 0.095 Lower limit of 95% CI 2.07 2.01 0.940 1.92 1.93 0.893 1.92 1.93 0.893 Upper limit of 95% CI 18.8 15.3 4.40 17.5 14.7 4.12 17.5 14.7 4.12

TABLE 13.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 31.3 111 31.3 111 31.3 111 Average 154 380 154 380 153 376 Stdev 345 636 345 636 349 629 p (t-test) 0.012 0.012 0.014 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 63 83 63 83 61 85 sCr only Median 32.9 110 32.9 110 32.1 111 Average 157 394 157 394 154 393 Stdev 339 652 339 652 340 648 p (t-test) 0.0082 0.0082 0.0075 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 67 78 67 78 66 79 UO only Median 61.7 121 61.7 121 63.7 119 Average 231 446 231 446 238 423 Stdev 398 762 398 762 404 745 p (t-test) 0.039 0.039 0.072 Min 0.259 3.55 0.259 3.55 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 80 48 80 48 77 51 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.69 0.64 0.69 0.69 0.64 0.69 0.69 0.61 SE 0.043 0.044 0.052 0.043 0.044 0.052 0.043 0.043 0.051 p Value 7.6E−6 2.0E−5 0.0079 7.6E−6 2.0E−5 0.0079 7.6E−6 7.0E−6 0.027 nCohort Recovered 63 67 80 63 67 80 61 66 77 nCohort Non-recovered 83 78 48 83 78 48 85 79 51 Cutoff Quartile 2 17.4 17.2 20.7 17.4 17.2 20.7 17.4 17.2 20.7 Sensitivity 84% 85% 85% 84% 85% 85% 84% 85% 82% Specificity 38% 36% 31% 38% 36% 31% 38% 36% 30% Cutoff Quartile 3 80.9 83.5 87.1 80.9 83.5 87.1 80.9 83.5 87.1 Sensitivity 64% 64% 60% 64% 64% 60% 64% 65% 59% Specificity 68% 66% 56% 68% 66% 56% 69% 67% 56% Cutoff Quartile 4 325 331 369 325 331 369 325 331 369 Sensitivity 34% 35% 31% 34% 35% 31% 34% 35% 29% Specificity 86% 85% 79% 86% 85% 79% 87% 86% 78% OR Quartile 2 3.31 3.07 2.66 3.31 3.07 2.66 3.07 3.19 1.99 p Value 0.0026 0.0055 0.039 0.0026 0.0055 0.039 0.0044 0.0041 0.12 Lower limit of 95% CI 1.52 1.39 1.05 1.52 1.39 1.05 1.42 1.44 0.833 Upper limit of 95% CI 7.23 6.78 6.75 7.23 6.78 6.75 6.65 7.05 4.74 OR Quartile 3 3.80 3.42 1.96 3.80 3.42 1.96 3.85 3.64 1.81 p Value 1.6E−4 4.3E−4 0.069 1.6E−4 4.3E−4 0.069 1.6E−4 2.3E−4 0.11 Lower limit of 95% CI 1.90 1.72 0.948 1.90 1.72 0.948 1.91 1.83 0.883 Upper limit of 95% CI 7.60 6.77 4.06 7.60 6.77 4.06 7.75 7.25 3.70 OR Quartile 4 3.05 3.02 1.68 3.05 3.02 1.68 3.43 3.48 1.47 p Value 0.0091 0.0081 0.21 0.0091 0.0081 0.21 0.0054 0.0037 0.35 Lower limit of 95% CI 1.32 1.33 0.748 1.32 1.33 0.748 1.44 1.50 0.656 Upper limit of 95% CI 7.07 6.84 3.79 7.07 6.84 3.79 8.17 8.06 3.30

TABLE 13.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts at 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 33.8 119 33.8 119 32.1 123 Average 144 432 145 416 136 413 Stdev 327 673 334 659 335 649 p (t-test) 0.0015 0.0029 0.0024 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 71 71 67 75 64 78 sCr only Median 36.8 119 35.3 123 33.8 126 Average 148 437 145 436 138 439 Stdev 325 681 327 677 323 672 p (t-test) 0.0015 0.0014 8.8E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 73 69 72 70 71 71 UO only Median 72.2 109 72.2 109 75.9 109 Average 259 422 249 415 258 406 Stdev 447 757 422 746 427 733 p (t-test) 0.14 0.13 0.17 Min 0.259 5.59 0.259 2.91 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 74 45 68 51 67 53 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.71 0.62 0.71 0.72 0.61 0.72 0.73 0.60 SE 0.043 0.043 0.054 0.043 0.043 0.053 0.042 0.042 0.052 p Value 3.4E−7 9.8E−7 0.032 1.0E−6 3.0E−7 0.046 8.3E−8 4.5E−8 0.061 nCohort Recovered 71 73 74 67 72 68 64 71 67 nCohort Non-recovered 71 69 45 75 70 51 78 71 53 Cutoff Quartile 2 17.4 17.4 25.6 17.4 17.4 25.6 17.4 17.4 25.8 Sensitivity 87% 87% 84% 85% 87% 82% 86% 87% 83% Specificity 38% 37% 31% 37% 38% 31% 39% 38% 31% Cutoff Quartile 3 80.9 80.9 87.9 80.9 80.9 87.9 80.9 80.9 90.6 Sensitivity 66% 67% 58% 65% 67% 57% 65% 68% 57% Specificity 66% 66% 54% 67% 67% 54% 69% 68% 55% Cutoff Quartile 4 332 332 364 332 332 364 332 332 369 Sensitivity 38% 38% 29% 37% 39% 27% 37% 39% 26% Specificity 87% 86% 77% 88% 88% 76% 89% 89% 76% OR Quartile 2 4.23 3.91 2.45 3.46 4.07 2.09 3.90 4.23 2.23 p Value 8.6E−4 0.0016 0.063 0.0026 0.0012 0.10 0.0010 8.6E−4 0.075 Lower limit of 95% CI 1.81 1.68 0.952 1.54 1.74 0.861 1.73 1.81 0.922 Upper limit of 95% CI 9.87 9.12 6.30 7.78 9.49 5.05 8.79 9.87 5.40 OR Quartile 3 3.84 3.84 1.61 3.85 4.09 1.57 4.16 4.36 1.61 p Value 1.5E−4 1.5E−4 0.21 1.5E−4 7.9E−5 0.22 7.5E−5 4.1E−5 0.20 Lower limit of 95% CI 1.91 1.91 0.762 1.92 2.03 0.757 2.05 2.16 0.778 Upper limit of 95% CI 7.69 7.70 3.40 7.74 8.22 3.27 8.41 8.80 3.32 OR Quartile 4 4.23 3.81 1.36 4.39 4.40 1.23 4.82 5.13 1.14 p Value 8.6E−4 0.0015 0.47 9.0E−4 6.2E−4 0.63 7.0E−4 2.6E−4 0.75 Lower limit of 95% CI 1.81 1.67 0.587 1.83 1.88 0.535 1.94 2.14 0.499 Upper limit of 95% CI 9.87 8.70 3.16 10.5 10.3 2.83 12.0 12.3 2.62

TABLE 13.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “recovered” and “non-recovered” cohorts where recovery starts within 7 days after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Recovery Period Duration (hr) 24 48 72 Recovered Non-recovered Recovered Non-recovered Recovered Non-recovered Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 40.8 118 44.2 117 40.8 118 Average 203 409 205 402 206 398 Stdev 383 708 384 703 386 698 p (t-test) 0.022 0.029 0.032 Min 0.259 5.59 0.259 2.91 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 93 56 92 57 91 58 sCr only Median 38.1 123 38.1 123 38.1 123 Average 149 446 149 446 149 446 Stdev 312 694 312 694 312 694 p (t-test) 6.5E−4 6.5E−4 6.5E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 83 66 83 66 83 66 UO only Median 58.9 118 58.9 118 58.9 118 Average 208 437 208 437 211 423 Stdev 373 757 373 757 377 745 p (t-test) 0.014 0.014 0.022 Min 0.259 3.55 0.259 3.55 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 100 48 100 48 98 50 Recovery Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.72 0.64 0.66 0.72 0.64 0.66 0.72 0.63 SE 0.047 0.043 0.050 0.047 0.043 0.050 0.047 0.043 0.050 p Value 2.1E−4 4.0E−7 0.0065 6.7E−4 4.0E−7 0.0065 4.5E−4 4.0E−7 0.012 nCohort Recovered 93 83 100 92 83 100 91 83 98 nCohort Non-recovered 56 66 48 57 66 48 58 66 50 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 88% 88% 85% 86% 88% 85% 86% 88% 84% Specificity 32% 35% 30% 32% 35% 30% 32% 35% 30% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 68% 68% 62% 67% 68% 62% 67% 68% 62% Specificity 60% 64% 56% 60% 64% 56% 60% 64% 56% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 34% 39% 33% 33% 39% 33% 33% 39% 32% Specificity 80% 86% 79% 79% 86% 79% 79% 86% 79% OR Quartile 2 3.33 3.89 2.51 2.82 3.89 2.51 2.92 3.89 2.21 p Value 0.0090 0.0021 0.047 0.019 0.0021 0.047 0.015 0.0021 0.075 Lower limit of 95% CI 1.35 1.64 1.01 1.18 1.64 1.01 1.23 1.64 0.923 Upper limit of 95% CI 8.23 9.26 6.23 6.71 9.26 6.23 6.96 9.26 5.28 OR Quartile 3 3.20 3.79 2.12 2.97 3.79 2.12 3.14 3.79 2.09 p Value 0.0011 1.4E−4 0.037 0.0020 1.4E−4 0.037 0.0012 1.4E−4 0.038 Lower limit of 95% CI 1.59 1.91 1.05 1.49 1.91 1.05 1.57 1.91 1.04 Upper limit of 95% CI 6.42 7.51 4.29 5.93 7.51 4.29 6.26 7.51 4.19 OR Quartile 4 2.00 3.85 1.88 1.92 3.85 1.88 1.85 3.85 1.73 p Value 0.069 7.8E−4 0.11 0.087 7.8E−4 0.11 0.11 7.8E−4 0.16 Lower limit of 95% CI 0.946 1.75 0.872 0.910 1.75 0.872 0.876 1.75 0.803 Upper limit of 95% CI 4.23 8.44 4.06 4.05 8.44 4.06 3.89 8.44 3.71

Example 14. Use of Insulin-Like Growth Factor-Binding Protein 2 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 2 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 14.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 3.47 15.6 3.49 15.6 3.53 11.3 Average 12.3 25.4 12.9 26.3 14.9 22.7 Stdev 21.5 31.8 22.2 32.4 25.0 29.1 p (t-test) 0.0033 0.0043 0.12 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 98 51 106 43 113 36 sCr only Median 3.47 14.5 3.52 15.6 3.55 11.3 Average 12.4 24.9 12.9 26.3 15.0 22.0 Stdev 21.6 31.7 22.2 32.4 25.2 28.6 p (t-test) 0.0049 0.0042 0.16 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 97 52 106 43 111 38 UO only Median 3.75 17.2 3.55 21.0 3.55 21.0 Average 15.9 23.7 15.8 24.9 15.8 24.9 Stdev 26.1 26.8 26.0 27.1 26.0 27.1 p (t-test) 0.24 0.18 0.18 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 130 18 131 17 131 17 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.62 0.68 0.65 0.63 0.70 0.61 0.59 0.70 SE 0.049 0.049 0.073 0.052 0.052 0.074 0.056 0.055 0.074 p Value 0.0050 0.012 0.012 0.0045 0.011 0.0078 0.044 0.098 0.0078 nCohort Non-persistent 98 97 130 106 106 131 113 111 131 nCohort Persistent 51 52 18 43 43 17 36 38 17 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 76% 75% 94% 79% 77% 94% 78% 74% 94% Specificity 26% 25% 28% 26% 25% 27% 26% 24% 27% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 65% 63% 78% 65% 63% 82% 64% 61% 82% Specificity 57% 57% 54% 56% 55% 54% 54% 53% 54% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 43% 42% 50% 44% 47% 53% 42% 42% 53% Specificity 84% 84% 78% 82% 83% 79% 80% 80% 79% OR Quartile 2 1.11 0.986 6.51 1.36 1.13 6.06 1.21 0.900 6.06 p Value 0.79 0.97 0.074 0.48 0.78 0.086 0.68 0.81 0.086 Lower limit of 95% CI 0.505 0.453 0.836 0.578 0.491 0.776 0.495 0.388 0.776 Upper limit of 95% CI 2.45 2.15 50.7 3.18 2.59 47.4 2.95 2.09 47.4 OR Quartile 3 2.44 2.27 4.08 2.34 2.04 5.52 2.08 1.74 5.52 p Value 0.012 0.020 0.018 0.023 0.055 0.0096 0.065 0.15 0.0096 Lower limit of 95% CI 1.21 1.14 1.28 1.12 0.986 1.51 0.957 0.822 1.51 Upper limit of 95% CI 4.92 4.55 13.1 4.89 4.22 20.1 4.50 3.68 20.1 OR Quartile 4 3.89 3.71 3.64 3.62 4.25 4.14 2.80 2.94 4.14 p Value 5.5E−4 8.2E−4 0.012 0.0012 3.0E−4 0.0074 0.012 0.0078 0.0074 Lower limit of 95% CI 1.80 1.72 1.32 1.66 1.94 1.46 1.25 1.33 1.46 Upper limit of 95% CI 8.40 8.00 10.0 7.91 9.32 11.7 6.25 6.52 11.7

TABLE 14.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 3.15 10.8 3.19 13.4 3.47 10.2 Average 12.5 23.4 12.9 24.2 15.1 20.7 Stdev 22.2 30.4 22.9 30.3 25.8 26.9 p (t-test) 0.013 0.012 0.23 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 91 58 98 51 105 44 sCr only Median 3.24 10.8 3.24 12.4 3.47 9.73 Average 12.6 23.3 13.0 23.8 15.3 19.9 Stdev 22.2 30.5 23.0 30.2 26.0 26.5 p (t-test) 0.014 0.016 0.32 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 91 58 97 52 103 46 UO only Median 3.55 11.3 3.53 12.4 3.53 12.4 Average 16.3 19.8 16.2 20.5 16.2 20.5 Stdev 26.7 23.9 26.6 24.2 26.6 24.2 p (t-test) 0.54 0.46 0.46 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 123 25 124 24 124 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.63 0.62 0.64 0.65 0.64 0.65 0.62 0.60 0.65 SE 0.047 0.048 0.064 0.049 0.049 0.065 0.052 0.051 0.065 p Value 0.0049 0.013 0.029 0.0014 0.0035 0.023 0.022 0.064 0.023 nCohort Non-persistent 91 91 123 98 97 124 105 103 124 nCohort Persistent 58 58 25 51 52 24 44 46 24 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 78% 76% 88% 80% 79% 88% 80% 76% 88% Specificity 26% 25% 28% 28% 27% 27% 27% 25% 27% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 66% 64% 72% 67% 65% 75% 66% 63% 75% Specificity 59% 58% 54% 58% 58% 55% 56% 55% 55% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 40% 40% 40% 43% 42% 42% 39% 37% 42% Specificity 84% 84% 78% 84% 84% 78% 80% 80% 78% OR Quartile 2 1.24 1.06 2.80 1.56 1.36 2.64 1.41 1.07 2.64 p Value 0.59 0.88 0.11 0.29 0.45 0.13 0.42 0.86 0.13 Lower limit of 95% CI 0.572 0.495 0.787 0.686 0.611 0.741 0.604 0.478 0.741 Upper limit of 95% CI 2.69 2.28 9.97 3.54 3.05 9.44 3.31 2.42 9.44 OR Quartile 3 2.77 2.46 3.08 2.78 2.58 3.64 2.48 2.11 3.64 p Value 0.0035 0.0094 0.019 0.0046 0.0079 0.010 0.015 0.040 0.010 Lower limit of 95% CI 1.40 1.25 1.20 1.37 1.28 1.35 1.19 1.04 1.35 Upper limit of 95% CI 5.50 4.84 7.89 5.64 5.19 9.80 5.16 4.32 9.80 OR Quartile 4 3.33 3.33 2.37 3.89 3.71 2.57 2.52 2.29 2.57 p Value 0.0020 0.0020 0.062 5.5E−4 8.2E−4 0.044 0.019 0.034 0.044 Lower limit of 95% CI 1.55 1.55 0.957 1.80 1.72 1.03 1.16 1.06 1.03 Upper limit of 95% CI 7.15 7.15 5.87 8.40 8.00 6.42 5.45 4.93 6.42

TABLE 14.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 3.09 10.3 2.97 13.4 3.19 10.3 Average 11.6 23.8 11.1 25.3 14.6 21.0 Stdev 20.2 31.4 19.9 31.9 25.4 27.4 p (t-test) 0.0044 0.0010 0.16 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 86 63 90 59 98 51 sCr only Median 3.15 9.73 3.09 11.3 3.47 8.85 Average 11.7 23.5 11.4 25.0 14.9 20.1 Stdev 20.3 31.3 19.9 32.0 25.4 27.4 p (t-test) 0.0062 0.0017 0.25 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 85 64 90 59 97 52 UO only Median 3.49 12.4 3.50 13.4 3.50 13.4 Average 15.7 21.2 15.6 21.8 15.6 21.8 Stdev 26.4 25.7 26.3 25.9 26.3 25.9 p (t-test) 0.29 0.24 0.24 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 116 32 117 31 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.63 0.65 0.68 0.66 0.66 0.63 0.60 0.66 SE 0.046 0.047 0.058 0.046 0.047 0.058 0.049 0.050 0.058 p Value 0.0027 0.0067 0.0089 1.1E−4 7.7E−4 0.0070 0.0087 0.047 0.0070 nCohort Non-persistent 86 85 116 90 90 117 98 97 117 nCohort Persistent 63 64 32 59 59 31 51 52 31 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 76% 75% 84% 80% 78% 84% 78% 75% 84% Specificity 26% 25% 28% 28% 27% 27% 27% 25% 27% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 65% 64% 72% 68% 66% 74% 67% 63% 74% Specificity 60% 60% 56% 61% 60% 56% 58% 57% 56% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 40% 39% 41% 42% 41% 42% 37% 35% 42% Specificity 85% 85% 79% 86% 84% 79% 81% 79% 79% OR Quartile 2 1.10 0.984 2.06 1.51 1.29 1.96 1.31 0.986 1.96 p Value 0.80 0.97 0.17 0.31 0.52 0.21 0.51 0.97 0.21 Lower limit of 95% CI 0.517 0.465 0.729 0.688 0.594 0.692 0.588 0.453 0.692 Upper limit of 95% CI 2.34 2.08 5.81 3.30 2.79 5.54 2.93 2.15 5.54 OR Quartile 3 2.85 2.67 3.26 3.31 2.92 3.72 2.78 2.27 3.72 p Value 0.0023 0.0040 0.0067 6.9E−4 0.0021 0.0036 0.0046 0.020 0.0036 Lower limit of 95% CI 1.45 1.37 1.39 1.66 1.48 1.54 1.37 1.14 1.54 Upper limit of 95% CI 5.60 5.23 7.65 6.60 5.80 9.00 5.64 4.55 9.00 OR Quartile 4 3.69 3.55 2.62 4.36 3.72 2.80 2.47 2.04 2.80 p Value 9.7E−4 0.0014 0.024 2.3E−4 8.4E−4 0.017 0.019 0.064 0.017 Lower limit of 95% CI 1.70 1.64 1.14 1.99 1.72 1.20 1.16 0.959 1.20 Upper limit of 95% CI 8.03 7.71 6.05 9.52 8.05 6.50 5.26 4.33 6.50

TABLE 14.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 3.03 10.8 2.97 13.4 3.19 10.3 Average 11.2 24.2 11.1 25.3 14.1 21.6 Stdev 20.0 31.3 19.9 31.9 24.9 27.9 p (t-test) 0.0024 0.0010 0.094 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 85 64 90 59 96 53 sCr only Median 3.15 9.73 3.03 12.4 3.24 9.73 Average 11.7 23.5 11.3 24.9 14.2 21.2 Stdev 20.3 31.3 20.0 31.8 25.0 27.8 p (t-test) 0.0062 0.0015 0.12 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 85 64 89 60 95 54 UO only Median 3.35 12.4 3.47 12.4 3.47 12.4 Average 14.8 23.3 15.0 23.2 15.0 23.2 Stdev 25.3 28.4 25.3 28.7 25.3 28.7 p (t-test) 0.090 0.11 0.11 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 112 36 114 34 114 34 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.65 0.63 0.67 0.68 0.66 0.67 0.63 0.62 0.67 SE 0.046 0.047 0.054 0.046 0.046 0.056 0.049 0.049 0.056 p Value 0.0011 0.0067 0.0017 1.1E−4 3.8E−4 0.0031 0.0067 0.016 0.0031 nCohort Non-persistent 85 85 112 90 89 114 96 95 114 nCohort Persistent 64 64 36 59 60 34 53 54 34 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 77% 75% 86% 80% 78% 85% 77% 76% 85% Specificity 26% 25% 29% 28% 27% 28% 26% 25% 28% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 66% 64% 72% 68% 67% 74% 66% 65% 74% Specificity 61% 60% 57% 61% 61% 57% 58% 58% 57% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 41% 39% 42% 42% 42% 41% 38% 37% 41% Specificity 86% 85% 80% 86% 85% 80% 81% 81% 80% OR Quartile 2 1.14 0.984 2.48 1.51 1.33 2.26 1.20 1.07 2.26 p Value 0.73 0.97 0.084 0.31 0.46 0.12 0.65 0.87 0.12 Lower limit of 95% CI 0.536 0.465 0.886 0.688 0.617 0.805 0.547 0.490 0.805 Upper limit of 95% CI 2.43 2.08 6.94 3.30 2.89 6.36 2.65 2.32 6.36 OR Quartile 3 3.01 2.67 3.47 3.31 3.09 3.68 2.72 2.53 3.68 p Value 0.0014 0.0040 0.0030 6.9E−4 0.0013 0.0026 0.0049 0.0084 0.0026 Lower limit of 95% CI 1.53 1.37 1.53 1.66 1.56 1.58 1.35 1.27 1.58 Upper limit of 95% CI 5.91 5.23 7.87 6.60 6.12 8.60 5.47 5.06 8.60 OR Quartile 4 4.16 3.55 2.92 4.36 4.18 2.77 2.63 2.52 2.77 p Value 3.9E−4 0.0014 0.0095 2.3E−4 3.3E−4 0.015 0.012 0.016 0.015 Lower limit of 95% CI 1.89 1.64 1.30 1.99 1.91 1.22 1.23 1.18 1.22 Upper limit of 95% CI 9.16 7.71 6.57 9.52 9.12 6.30 5.59 5.35 6.30

TABLE 14.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.97 11.3 2.85 15.6 3.15 10.8 Average 10.3 24.7 9.85 26.2 13.9 21.6 Stdev 18.3 31.8 17.9 32.2 25.0 27.5 p (t-test) 6.6E−4 1.2E−4 0.081 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 82 67 86 63 93 56 sCr only Median 3.03 10.8 2.91 14.5 3.15 10.8 Average 10.7 24.4 10.3 25.9 13.4 22.4 Stdev 18.6 31.9 18.2 32.4 23.9 28.9 p (t-test) 0.0013 2.6E−4 0.042 Min 3.16E−5 0.0532 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 83 66 87 62 93 56 UO only Median 3.24 13.4 3.35 14.5 3.35 14.5 Average 14.7 23.2 14.6 23.8 14.6 23.8 Stdev 25.4 28.0 25.3 28.2 25.3 28.2 p (t-test) 0.089 0.069 0.069 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 111 112 111 112 111 112 n (Patient) 111 37 112 36 112 36 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.65 0.64 0.68 0.69 0.68 0.68 0.63 0.64 0.68 SE 0.045 0.046 0.054 0.045 0.045 0.054 0.048 0.048 0.054 p Value 6.8E−4 0.0017 9.7E−4 1.8E−5 6.2E−5 7.3E−4 0.0052 0.0047 7.3E−4 nCohort Non-persistent 82 83 111 86 87 112 9.3 93 112 nCohort Persistent 67 66 37 63 62 36 56 56 36 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 76% 76% 86% 79% 79% 86% 77% 77% 86% Specificity 26% 25% 29% 28% 28% 29% 26% 26% 29% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 66% 65% 73% 68% 68% 75% 66% 66% 75% Specificity 62% 61% 58% 63% 62% 58% 59% 59% 58% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 40% 39% 41% 43% 42% 42% 38% 38% 42% Specificity 87% 86% 80% 87% 86% 80% 82% 82% 80% OR Quartile 2 1.10 1.06 2.59 1.49 1.44 2.48 1.15 1.15 2.48 p Value 0.81 0.88 0.069 0.31 0.36 0.084 0.72 0.72 0.084 Lower limit of 95% CI 0.519 0.500 0.927 0.689 0.664 0.886 0.530 0.530 0.886 Upper limit of 95% CI 2.32 2.24 7.25 3.22 3.11 6.94 2.50 2.50 6.94 OR Quartile 3 3.15 2.98 3.68 3.63 3.44 4.15 2.82 2.82 4.15 p Value 8.5E−4 0.0015 0.0018 2.4E−4 4.2E−4 9.3E−4 0.0033 0.0033 9.3E−4 Lower limit of 95% CI 1.60 1.52 1.62 1.82 1.73 1.79 1.41 1.41 1.79 Upper limit of 95% CI 6.17 5.84 8.33 7.22 6.83 9.64 5.62 5.62 9.64 OR Quartile 4 4.36 3.85 2.76 5.11 4.51 2.92 2.68 2.68 2.92 p Value 3.2E−4 7.8E−4 0.014 7.2E−5 1.9E−4 0.0095 0.010 0.010 0.0095 Lower limit of 95% CI 1.96 1.75 1.23 2.28 2.05 1.30 1.26 1.26 1.30 Upper limit of 95% CI 9.71 8.44 6.17 11.4 9.96 6.57 5.70 5.70 6.57

Example 15. Use of Insulin-Like Growth Factor-Binding Protein 2 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 2 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 15.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.31 9.57 2.97 7.04 2.97 11.3 Average 6.67 22.7 9.93 24.3 9.33 27.5 Stdev 12.6 30.0 14.8 33.1 14.1 34.7 D (t-test) 2.5E−4 7.0E−4 1.9E−5 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 55 94 78 71 88 61 sCr only Median 2.35 9.57 2.97 7.04 3.03 10.8 Average 7.38 22.6 10.3 23.5 9.80 27.1 Stdev 13.4 30.2 15.4 32.7 14.6 34.9 p (t-test) 4.7E−4 0.0019 5.2E−5 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 57 92 76 73 89 60 UO only Median 3.75 6.98 3.96 13.4 3.53 22.0 Average 15.1 22.7 14.7 26.4 14.3 30.6 Stdev 24.7 30.4 24.2 32.6 23.9 33.7 p (t-test) 0.14 0.037 0.0060 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 114 34 121 27 125 23 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.69 0.61 0.63 0.62 0.64 0.66 0.65 0.71 SE 0.042 0.043 0.057 0.046 0.046 0.062 0.046 0.047 0.064 p Value 5.6E−7 1.7E−5 0.050 0.0050 0.0092 0.020 4.7E−4 0.0013 0.0014 nCohort Non-persistent 55 57 114 78 76 121 88 89 125 nCohort Persistent 94 92 34 71 73 27 61 60 23 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 81% 79% 82% 77% 77% 81% 79% 78% 87% Specificity 35% 32% 27% 27% 26% 26% 27% 27% 27% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 63% 62% 62% 59% 60% 67% 64% 65% 78% Specificity 71% 68% 54% 58% 59% 54% 59% 60% 55% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 37% 37% 41% 37% 36% 48% 43% 42% 57% Specificity 95% 93% 80% 85% 84% 80% 86% 85% 81% OR Quartile 2 2.23 1.77 1.74 1.27 1.18 1.58 1.38 1.33 2.49 p Value 0.038 0.14 0.26 0.54 0.67 0.39 0.41 0.46 0.16 Lower limit of 95% CI 1.05 0.835 0.658 0.599 0.558 0.553 0.640 0.617 0.695 Upper limit of 95% CI 4.75 3.76 4.61 2.68 2.48 4.53 3.00 2.89 8.92 OR Quartile 3 4.11 3.53 1.86 1.97 2.20 2.32 2.56 2.73 4.44 p Value 1.1E−4 4.1E−4 0.12 0.041 0.018 0.060 0.0062 0.0037 0.0055 Lower limit of 95% CI 2.01 1.75 0.849 1.03 1.14 0.966 1.31 1.39 1.55 Upper limit of 95% CI 8.41 7.10 4.07 3.79 4.24 5.58 5.02 5.39 12.7 OR Quartile 4 10.3 7.77 2.77 3.18 2.95 3.75 4.70 4.18 5.47 p Value 2.2E−4 2.6E−4 0.015 0.0038 0.0066 0.0031 1.3E−4 3.3E−4 3.8E−4 Lower limit of 95% CI 2.99 2.58 1.22 1.45 1.35 1.56 2.13 1.91 2.14 Upper limit of 95% CI 35.4 23.4 6.30 6.95 6.44 9.02 10.4 9.12 14.0

TABLE 15.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.30 9.12 2.65 8.16 2.61 11.3 Average 6.65 22.5 9.62 23.6 8.94 26.3 Stdev 12.7 29.9 15.1 32.2 14.4 33.3 p (t-test) 3.0E−4 9.4E−4 3.6E−5 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 54 95 73 76 82 67 sCr only Median 2.35 9.12 2.79 7.39 2.72 10.3 Average 7.37 22.4 10.3 2.3.0 9.59 25.5 Stdev 13.6 30.1 15.7 32.2 15.0 33.4 p (t-test) 5.6E−4 0.0026 1.6E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 56 93 73 76 82 67 UO only Median 3.49 7.04 3.51 13.4 3.47 21.0 Average 14.5 23.0 14.1 25.9 13.7 28.9 Stdev 24.0 30.5 23.5 32.3 23.2 33.1 p (t-test) 0.075 0.020 0.0038 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 106 42 113 35 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.69 0.63 0.65 0.63 0.66 0.69 0.66 0.71 SE 0.042 0.043 0.052 0.045 0.045 0.055 0.044 0.045 0.057 p Value 2.8E−7 1.1E−5 0.012 7.2E−4 0.0048 0.0044 2.5E−5 3.9E−4 2.7E−4 nCohort Non-persistent 54 56 106 73 73 113 82 82 117 nCohort Persistent 95 93 42 76 76 35 67 67 31 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 81% 80% 83% 79% 78% 83% 81% 79% 87% Specificity 35% 32% 28% 29% 27% 27% 29% 28% 28% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 63% 62% 64% 62% 61% 69% 67% 66% 77% Specificity 72% 70% 56% 62% 60% 56% 63% 62% 57% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 37% 37% 40% 37% 36% 46% 42% 40% 52% Specificity 94% 93% 81% 86% 85% 81% 88% 87% 82% OR Quartile 2 2.32 1.84 1.97 1.51 1.31 1.83 1.72 1.48 2.65 p Value 0.029 0.11 0.15 0.28 0.48 0.22 0.17 0.32 0.089 Lower limit of 95% CI 1.09 0.868 0.791 0.716 0.621 0.692 0.796 0.690 0.861 Upper limit of 95% CI 4.96 3.92 4.93 3.20 2.76 4.83 3.71 3.16 8.17 OR Quartile 3 4.46 3.80 2.26 2.60 2.33 2.75 3.55 3.15 4.59 p Value 5.6E−5 2.2E−4 0.030 0.0045 0.012 0.014 2.6E−4 8.5E−4 0.0011 Lower limit of 95% CI 2.15 1.87 1.08 1.35 1.21 1.23 1.80 1.60 1.83 Upper limit of 95% CI 9.22 7.71 4.73 5.04 4.49 6.15 7.00 6.17 11.5 OR Quartile 4 9.92 7.49 2.92 3.68 3.11 3.69 5.17 4.36 4.88 p Value 2.8E−4 3.4E−4 0.0074 0.0017 0.0052 0.0017 8.7E−5 3.2E−4 2.5E−4 Lower limit of 95% CI 2.88 2.49 1.33 1.63 1.40 1.63 2.28 1.96 2.09 Upper limit of 95% CI 34.2 22.5 6.41 8.29 6.88 8.35 11.7 9.71 11.4

TABLE 15.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.30 9.12 2.72 7.74 2.72 10.3 Average 6.65 22.5 9.72 23.3 9.19 25.1 Stdev 12.7 29.9 15.2 32.0 14.7 32.8 p (t-test) 3.0E−4 0.0013 1.6E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 54 95 72 77 78 71 sCr only Median 2.35 9.12 2.85 7.04 2.85 9.12 Average 7.37 22.4 10.4 22.7 9.79 24.4 Stdev 13.6 30.1 15.7 32.0 15.3 32.8 p (t-test) 5.6E−4 0.0036 5.3E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 56 93 72 77 78 71 UO only Median 3.49 7.04 3.49 10.8 3.47 14.5 Average 14.9 20.9 14.3 23.4 13.8 25.6 Stdev 24.6 29.1 24.1 30.3 23.7 31.1 p (t-test) 0.19 0.056 0.016 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 100 48 106 42 110 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.69 0.61 0.65 0.62 0.64 0.67 0.64 0.68 SE 0.042 0.043 0.051 0.045 0.046 0.052 0.044 0.045 0.053 p Value 2.8E−7 1.1E−5 0.031 0.0011 0.0069 0.0083 1.6E−4 0.0014 9.3E−4 nCohort Non-persistent 54 56 100 72 72 106 78 78 110 nCohort Persistent 95 93 48 77 77 42 71 71 38 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 81% 80% 79% 79% 78% 79% 79% 77% 82% Specificity 35% 32% 27% 29% 28% 26% 28% 27% 27% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 63% 62% 62% 61% 60% 67% 65% 63% 74% Specificity 72% 70% 56% 61% 60% 57% 63% 62% 58% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 37% 37% 35% 36% 35% 40% 39% 38% 45% Specificity 94% 93% 80% 86% 85% 81% 87% 86% 82% OR Quartile 2 2.32 1.84 1.41 1.57 1.36 1.32 1.47 1.27 1.66 p Value 0.029 0.11 0.42 0.24 0.42 0.53 0.32 0.54 0.28 Lower limit of 95% CI 1.09 0.868 0.616 0.742 0.644 0.560 0.690 0.599 0.661 Upper limit of 95% CI 4.96 3.92 3.21 3.32 2.86 3.09 3.12 2.68 4.17 OR Quartile 3 4.46 3.80 2.12 2.46 2.20 2.61 3.11 2.77 3.90 p Value 5.6E−5 2.2E−4 0.037 0.0074 0.018 0.012 9.0E−4 0.0027 0.0011 Lower limit of 95% CI 2.15 1.87 1.05 1.27 1.14 1.24 1.59 1.43 1.72 Upper limit of 95% CI 9.22 7.71 4.29 4.76 4.24 5.51 6.07 5.38 8.80 OR Quartile 4 9.92 7.49 2.19 3.54 2.99 2.92 4.43 3.74 3.64 p Value 2.8E−4 3.4E−4 0.045 0.0023 0.0068 0.0074 3.6E−4 0.0012 0.0016 Lower limit of 95% CI 2.88 2.49 1.02 1.57 1.35 1.33 1.96 1.68 1.63 Upper limit of 95% CI 34.2 22.5 4.73 7.99 6.63 6.41 10.0 8.30 8.13

TABLE 15.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.30 9.12 2.72 7.74 2.72 10.3 Average 6.65 22.5 9.72 23.3 9.19 25.1 Stdev 12.7 29.9 15.2 32.0 14.7 32.8 p (t-test) 3.0E−4 0.0013 1.6E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 54 95 72 77 78 71 sCr only Median 2.35 9.12 2.85 7.04 2.85 9.12 Average 7.37 22.4 10.4 22.7 9.79 24.4 Stdev 13.6 30.1 15.7 32.0 15.3 32.8 p (t-test) 5.6E−4 0.0036 5.3E−4 Min 3.16E−5 0.0532 3.16E−5 0.05.32 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 56 93 72 77 78 71 UO only Median 3.49 6.98 3.47 8.58 3.47 11.3 Average 15.1 20.3 14.6 22.1 14.1 24.0 Stdev 24.8 28.7 24.4 29.7 24.0 30.4 p (t-test) 0.26 0.11 0.039 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 98 50 103 45 107 41 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.69 0.60 0.65 0.62 0.62 0.67 0.64 0.66 SE 0.042 0.043 0.050 0.045 0.046 0.051 0.044 0.045 0.052 p Value 2.8E−7 1.1E−5 0.047 0.0011 0.0069 0.016 1.6E−4 0.0014 0.0023 nCohort Non-persistent 54 56 98 72 72 103 78 78 107 nCohort Persistent 95 93 50 77 77 45 71 71 41 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 81% 80% 78% 79% 78% 78% 79% 77% 80% Specificity 35% 32% 27% 29% 28% 26% 28% 27% 27% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 63% 62% 62% 61% 60% 64% 65% 63% 71% Specificity 72% 70% 56% 61% 60% 56% 63% 62% 58% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 37% 37% 34% 36% 35% 38% 39% 38% 41% Specificity 94% 93% 80% 86% 85% 81% 87% 86% 81% OR Quartile 2 2.32 1.84 1.28 1.57 1.36 1.24 1.47 1.27 1.53 p Value 0.029 0.11 0.55 0.24 0.42 0.61 0.32 0.54 0.34 Lower limit of 95% CI 1.09 0.868 0.572 0.742 0.644 0.543 0.690 0.599 0.635 Upper limit of 95% CI 4.96 3.92 2.87 3.32 2.86 2.85 3.12 2.68 3.71 OR Quartile 3 4.46 3.80 2.09 2.46 2.20 2.34 3.11 2.77 3.33 p Value 5.6E−5 2.2E−4 0.038 0.0074 0.018 0.022 9.0E−4 0.0027 0.0023 Lower limit of 95% CI 2.15 1.87 1.04 1.27 1.14 1.13 1.59 1.43 1.53 Upper limit of 95% CI 9.22 7.71 4.19 4.76 4.24 4.82 6.07 5.38 7.22 OR Quartile 4 9.92 7.49 2.01 3.54 2.99 2.52 4.43 3.74 3.08 p Value 2.8E−4 3.4E−4 0.073 0.0023 0.0068 0.020 3.6E−4 0.0012 0.0052 Lower limit of 95% CI 2.88 2.49 0.936 1.57 1.35 1.16 1.96 1.68 1.40 Upper limit of 95% CI 34.2 22.5 4.31 7.99 6.63 5.47 10.0 8.30 6.78

TABLE 15.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 2.30 9.12 2.65 7.39 2.72 9.12 Average 6.53 22.2 9.85 22.7 9.38 24.0 Stdev 12.8 29.7 15.4 31.6 15.0 32.2 p (t-test) 3.8E−4 0.0025 5.1E−4 Mill 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 52 97 69 80 74 75 sCr only Median 2.34 9.57 2.72 7.74 2.72 10.3 Average 7.17 22.4 10.1 22.6 9.57 24.2 Stdev 13.6 29.9 15.9 31.6 15.4 32.4 p (t-test) 5.1E−4 0.0034 5.1E−4 Min 3.16E−5 0.0532 3.16E−5 0.0532 3.16E−5 0.0532 Max 78.7 112 78.7 112 78.7 112 n (Patient) 55 94 70 79 76 73 UO only Median 3.47 7.04 3.47 8.58 3.47 10.8 Average 15.1 20.2 14.7 21.2 14.4 22.4 Stdev 25.1 28.1 24.8 28.7 24.5 29.2 p (t-test) 0.26 0.16 0.084 Min 3.16E−5 0.0645 3.16E−5 0.0645 3.16E−5 0.0645 Max 109 112 109 112 109 112 n (Patient) 96 52 99 49 102 46 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.70 0.61 0.64 0.64 0.63 0.65 0.66 0.65 SE 0.042 0.043 0.049 0.045 0.045 0.050 0.045 0.045 0.050 p Value 4.8E−7 3.4E−6 0.024 0.0022 0.0026 0.012 5.3E−4 4.3E−4 0.0025 nCohort Non-persistent 52 55 96 69 70 99 74 76 102 nCohort Persistent 97 94 52 80 79 49 75 73 46 Cutoff Quartile 2 1.89 1.89 1.93 1.89 1.89 1.93 1.89 1.89 1.93 Sensitivity 80% 80% 79% 79% 78% 80% 79% 78% 83% Specificity 35% 33% 27% 29% 29% 27% 28% 28% 28% Cutoff Quartile 3 4.13 4.13 4.52 4.13 4.13 4.52 4.13 4.13 4.52 Sensitivity 63% 63% 63% 60% 61% 63% 63% 64% 67% Specificity 73% 71% 57% 61% 61% 57% 62% 63% 58% Cutoff Quartile 4 20.0 20.0 20.2 20.0 20.0 20.2 20.0 20.0 20.2 Sensitivity 36% 36% 33% 35% 34% 35% 37% 37% 37% Specificity 94% 93% 79% 86% 84% 80% 86% 86% 80% OR Quartile 2 2.17 1.92 1.38 1.51 1.46 1.46 1.46 1.36 1.89 p Value 0.045 0.090 0.43 0.28 0.32 0.37 0.32 0.42 0.16 Lower limit of 95% CI 1.02 0.902 0.620 0.717 0.692 0.642 0.691 0.643 0.786 Upper limit of 95% CI 4.65 4.09 3.09 3.19 3.08 3.33 3.09 2.88 4.53 OR Quartile 3 4.60 4.11 2.33 2.33 2.47 2.24 2.76 3.10 2.84 p Value 5.1E−5 1.1E−4 0.017 0.012 0.0074 0.024 0.0027 9.1E−4 0.0052 Lower limit of 95% CI 2.20 2.01 1.16 1.21 1.27 1.11 1.42 1.59 1.37 Upper limit of 95% CI 9.62 8.41 4.67 4.51 4.77 4.53 5.35 6.05 5.89 OR Quartile 4 9.22 7.22 1.85 3.18 2.78 2.10 3.81 3.47 2.40 p Value 4.3E−4 4.3E−4 0.11 0.0053 0.011 0.058 0.0013 0.0022 0.026 Lower limit of 95% CI 2.68 2.40 0.863 1.41 1.26 0.976 1.69 1.56 1.11 Upper limit of 95% CI 31.8 21.7 3.95 7.16 6.16 4.51 8.61 7.69 5.21

Example 16. Use of Insulin-Like Growth Factor-Binding Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 16.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 637 1250 637 1350 702 1160 Average 1820 4620 1960 4820 2360 4100 Stdev 3970 6250 4060 6480 4660 5940 p (t-test) 0.0011 0.0015 0.072 Min 20.6 158 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 98 51 106 43 113 36 sCr only Median 640 1210 641 1400 702 1160 Average 1840 4540 1840 5110 2280 4250 Stdev 3990 6220 3880 6620 4560 6050 p (t-test) 0.0016 2.6E−4 0.037 Min 20.6 158 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 97 52 106 43 111 38 UO only Median 747 1.380 766 1400 766 1400 Average 2440 5410 2430 5670 2430 5670 Stdev 4550 7420 4530 7560 4530 7560 p (t-test) 0.019 0.012 0.012 Min 20.6 322 20.6 322 20.6 322 Max 18800 18800 18800 18800 18800 18800 n (Patient) 130 18 131 17 131 17 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.67 0.70 0.70 0.69 0.70 0.65 0.64 0.70 SE 0.048 0.048 0.072 0.050 0.050 0.074 0.055 0.054 0.074 p Value 1.3E−4 4.6E−4 0.0058 7.9E−5 1.1E−4 0.0057 0.0048 0.0098 0.0057 nCohort Non-persistent 98 97 130 106 106 131 113 111 131 nCohort Persistent 51 52 18 43 43 17 36 38 17 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 84% 83% 89% 86% 84% 88% 86% 82% 88% Specificity 30% 29% 27% 29% 28% 27% 28% 27% 27% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 67% 65% 78% 72% 72% 76% 69% 68% 76% Specificity 58% 58% 54% 58% 58% 53% 56% 56% 53% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 41% 40% 33% 42% 44% 35% 36% 37% 35% Specificity 83% 82% 76% 81% 82% 76% 78% 78% 76% OR Quartile 2 2.26 1.94 2.95 2.55 2.03 2.73 2.45 1.64 2.73 p Value 0.067 0.12 0.16 0.056 0.13 0.20 0.088 0.29 0.20 Lower limit of 95% CI 0.946 0.835 0.644 0.977 0.814 0.595 0.875 0.653 0.595 Upper limit of 95% CI 5.39 4.50 13.5 6.65 5.06 12.6 6.86 4.12 12.6 OR Quartile 3 2.78 2.58 4.08 3.64 3.64 3.73 2.86 2.74 3.73 p Value 0.0046 0.0079 0.018 0.0010 0.0010 0.028 0.0100 0.011 0.028 Lower limit of 95% CI 1.37 1.28 1.28 1.68 1.68 1.16 1.29 1.26 1.16 Upper limit of 95% CI 5.64 5.19 13.1 7.86 7.86 12.0 6.38 5.98 12.0 OR Quartile 4 3.34 3.19 1.60 3.10 3.62 1.76 1.99 2.11 1.76 p Value 0.0020 0.0029 0.39 0.0044 0.0012 0.30 0.097 0.066 0.30 Lower limit of 95% CI 1.55 1.49 0.553 1.42 1.66 0.602 0.883 0.951 0.602 Upper limit of 95% CI 7.16 6.83 4.61 6.73 7.91 5.15 4.48 4.70 5.15

TABLE 16.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 626 1210 624 1400 640 1210 Average 1880 4200 1850 4570 2310 3920 Stdev 4110 5970 4030 6210 4680 5690 p (t-test) 0.0055 0.0015 0.074 Min 21.1 20.6 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 91 58 98 51 105 44 sCr only Median 634 1120 626 1300 642 1070 Average 1900 4170 1880 4470 2360 3740 Stdev 4110 5990 4040 6190 4710 5630 p (t-test) 0.0067 0.0025 0.12 Min 21.1 20.6 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 91 58 97 52 103 46 UO only Median 718 1350 724 1380 724 1380 Average 2490 4330 2480 4470 2480 4470 Stdev 4660 6520 4640 6630 4640 6630 p (t-test) 0.097 0.076 0.076 Min 20.6 322 20.6 322 20.6 322 Max 18800 18800 18800 18800 18800 18800 n (Patient) 193 25 124 24 124 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.66 0.68 0.71 0.70 0.69 0.67 0.64 0.69 SE 0.046 0.047 0.063 0.046 0.047 0.064 0.051 0.051 0.064 p Value 1.2E−4 7.6E−4 0.0036 3.7E−6 3.0E−5 0.0037 8.3E−4 0.0069 0.0037 nCohort Non-persistent 91 91 123 98 97 124 105 103 124 nCohort Persistent 58 58 25 51 52 24 44 46 24 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 84% 83% 92% 88% 87% 92% 89% 85% 92% Specificity 31% 30% 28% 32% 31% 28% 30% 29% 28% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 66% 64% 76% 71% 69% 75% 68% 65% 75% Specificity 59% 58% 55% 60% 60% 55% 57% 56% 55% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 40% 38% 32% 43% 40% 33% 36% 33% 33% Specificity 84% 82% 76% 84% 82% 77% 79% 78% 77% OR Quartile 2 2.42 2.02 4.57 3.47 2.88 4.33 3.42 2.29 4.33 p Value 0.039 0.090 0.047 0.010 0.022 0.056 0.018 0.074 0.056 Lower limit of 95% CI 1.05 0.895 1.02 1.34 1.16 0.966 1.23 0.922 0.966 Upper limit of 95% CI 5.60 4.58 20.4 8.99 7.12 19.4 9.48 5.69 19.4 OR Quartile 3 2.77 2.46 3.92 3.63 3.35 3.64 2.86 2.42 3.64 p Value 0.0035 0.0094 0.0066 5.0E−4 9.3E−4 0.010 0.0056 0.016 0.010 Lower limit of 95% CI 1.40 1.25 1.46 1.76 1.64 1.35 1.36 1.18 1.35 Upper limit of 95% CI 5.50 4.84 10.5 7.50 6.84 9.80 6.01 4.97 9.80 OR Quartile 4 3.33 2.86 1.53 3.89 3.19 1.64 2.16 1.68 1.64 p Value 0.0020 0.0064 0.38 5.5E−4 0.0029 0.31 0.052 0.19 0.31 Lower limit of 95% CI 1.55 1.34 0.597 1.80 1.49 0.6.37 0.995 0.778 0.6.37 Upper limit of 95% CI 7.15 6.11 3.90 8.40 6.83 4.21 4.67 3.64 4.21

TABLE 16.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 624 1070 608 1250 630 1070 Average 1750 4190 1680 4460 2290 3720 Stdev 3830 6070 3760 6180 4690 5570 p (t-test) 0.0031 8.3E−4 0.099 Min 21.1 20.6 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 86 63 90 59 98 51 sCr only Median 626 1070 619 1250 642 1070 Average 1770 4130 1690 4450 2320 3650 Stdev 3850 6040 3750 6190 4700 5540 p (t-test) 0.0042 9.1E−4 0.13 Min 21.1 20.6 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 85 64 90 59 97 52 UO only Median 724 1210 729 1250 729 1250 Average 2450 4070 2430 4170 2430 4170 Stdev 4660 6160 4640 6240 4640 6240 p (t-test) 0.11 0.088 0.088 Min 20.6 233 20.6 233 20.6 233 Max 18800 18800 18800 18800 18800 18800 n (Patient) 116 32 117 31 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.67 0.66 0.65 0.71 0.70 0.66 0.65 0.63 0.66 SE 0.045 0.046 0.058 0.044 0.045 0.058 0.049 0.049 0.058 p Value 2.2E−4 6.9E−4 0.0071 2.4E−6 1.3E−5 0.0077 0.0018 0.0093 0.0077 nCohort Non-persistent 86 85 116 90 90 117 98 97 117 nCohort Persistent 63 64 32 59 59 31 51 52 31 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 84% 83% 91% 88% 86% 90% 88% 85% 90% Specificity 31% 31% 29% 33% 32% 29% 32% 30% 29% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 63% 62% 69% 68% 66% 68% 65% 62% 68% Specificity 59% 59% 55% 61% 60% 55% 57% 56% 55% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 38% 38% 31% 41% 41% 32% 33% 33% 32% Specificity 84% 84% 77% 84% 84% 77% 79% 78% 77% OR Quartile 2 2.43 2.12 4.01 3.71 3.03 3.82 3.47 2.35 3.82 p Value 0.033 0.064 0.030 0.0044 0.012 0.036 0.010 0.055 0.036 Lower limit of 95% CI 1.07 0.957 1.14 1.51 1.27 1.09 1.34 0.983 1.09 Upper limit of 95% CI 5.48 4.71 14.0 9.16 7.21 13.4 8.99 5.60 13.4 OR Quartile 3 2.53 2.38 2.71 3.31 2.92 2.54 2.44 2.01 2.54 p Value 0.0065 0.011 0.019 6.9E−4 0.0021 0.029 0.012 0.047 0.029 Lower limit of 95% CI 1.30 1.22 1.18 1.66 1.48 1.10 1.21 1.01 1.10 Upper limit of 95% Cl 4.95 4.63 6.22 6.60 5.80 5.85 4.92 4.00 5.85 OR Quartile 4 3.16 3.04 1.50 3.72 3.72 1.59 1.83 1.76 1.59 p Value 0.0032 0.0043 0.36 8.4E−4 8.4E−4 0.30 0.12 0.14 0.30 Lower limit of 95% CI 1.47 1.42 0.632 1.72 1.72 0.667 0.861 0.827 0.667 Upper limit of 95% CI 6.81 6.54 3.55 8.05 8.05 3.78 3.91 3.74 3.78

TABLE 16.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 622 1120 605 1250 630 1070 Average 1550 4420 1680 4460 2140 3940 Stdev 3370 6290 3760 6180 4420 5860 p (t-test) 4.6E−4 8.3E−4 0.0.36 Min 21.1 20.6 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 85 64 90 59 96 53 sCr only Median 626 1070 615 1210 634 1070 Average 1770 4130 1700 4390 2160 3870 Stdev 3850 6040 3770 6150 4430 5830 p (t-test) 0.0042 0.0012 0.046 Min 21.1 20.6 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 85 64 89 60 95 54 UO only Median 724 1210 747 1210 747 1210 Average 2220 4590 2360 4280 2360 4280 Stdev 4.310 6610 4540 6310 4540 6310 p (t-test) 0.014 0.050 0.050 Min 20.6 233 20.6 233 20.6 233 Max 18800 18800 18800 18800 18800 18800 n (Patient) 112 36 114 34 114 34 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUG 0.68 0.66 0.66 0.71 0.70 0.65 0.65 0.64 0.65 SE 0.045 0.046 0.055 0.044 0.045 0.056 0.048 0.048 0.056 p Value 5.9E−5 6.9E−4 0.0026 2.4E−6 1.2E−5 0.0076 0.0013 0.0036 0.0076 nCohort Non-persistent 85 85 112 90 89 114 96 95 114 nCohort Persistent 64 64 36 59 60 34 53 54 34 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 84% 83% 92% 88% 87% 91% 87% 85% 91% Specificity 32% 31% 30% 33% 33% 30% 31% 31% 30% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 64% 62% 67% 68% 67% 65% 64% 63% 65% Specificity 60% 59% 55% 61% 61% 54% 57% 57% 54% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 39% 38% 33% 41% 40% 32% 34% 33% 32% Specificity 85% 84% 78% 84% 84% 77% 79% 79% 77% OR Quartile 2 2.51 2.12 4.79 3.71 3.14 4.39 2.99 2.53 4.39 p Value 0.027 0.064 0.014 0.0044 0.0096 0.020 0.018 0.036 0.020 Lower limit of 95% Cl 1.11 0.957 1.38 1.51 1.32 1.26 1.21 1.06 1.26 Upper limit of 95% CI 5.68 4.71 16.7 9.16 7.47 15.3 7.38 6.02 15.3 OR Quartile 3 2.67 2.38 2.48 3.31 3.09 2.19 2.40 2.24 2.19 p Value 0.0040 0.011 0.024 6.9E−4 0.0013 0.054 0.013 0.021 0.054 Lower limit of 95% CI 1.37 1.22 1.13 1.66 1.56 0.988 1.20 1.13 0.988 Upper limit of 95% CI 5.23 4.63 5.45 6.60 6.12 4.84 4.80 4.44 4.84 OR Quartile 4 3.55 3.04 1.74 3.72 3.57 1.62 1.95 1.88 1.62 p Value 0.0014 0.0043 0.19 8.4E−4 0.0012 0.26 0.081 0.10 0.26 Lower limit of 95% Cl 1.64 1.42 0.764 1.72 1.65 0.698 0.921 0.885 0.698 Upper limit of 95% CI 7.71 6.54 3.96 8.05 7.71 3.75 4.15 3.97 3.75

TABLE 16.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 611 1180 592 1350 626 1070 Average 1220 4690 1180 4970 1870 4290 Stdev 2550 6480 2500 6590 3980 6160 p (t-test) 1.7E−5 2.6E−6 0.0041 Min 21.1 20.6 20.6 158 20.6 158 Max 16600 18800 16600 18800 18800 18800 n (Patient) 82 67 86 63 93 56 sCr only Median 622 1120 601 1300 626 1070 Average 1430 4480 1380 4750 1870 4290 Stdev 3190 6290 3120 6400 3980 6160 p (t-test) 1.8E−4 3.6E−5 0.0041 Min 21.1 20.6 20.6 158 20.6 158 Max 18800 18800 18800 18800 18800 18800 n (Patient) 83 66 87 62 93 56 UO only Median 718 1180 724 1210 724 1210 Average 2230 4500 2220 4600 2220 4600 Stdev 4320 6540 4310 6610 4310 6610 p (t-test) 0.018 0.014 0.014 Min 20.6 233 20.6 233 20.6 233 Max 18800 18800 18800 18800 18800 18800 n (Patient) 111 37 112 36 112 36 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.68 0.67 0.73 0.72 0.67 0.66 0.66 0.67 SE 0.044 0.045 0.054 0.043 0.043 0.055 0.047 0.047 0.055 p Value 1.5E−5 6.5E−5 0.0021 5.9E−8 4.6E−7 0.0023 5.1E−4 5.1E−4 0.0023 nCohort Non-persistent 82 83 111 86 87 112 93 93 112 nCohort Persistent 67 66 37 63 62 36 56 56 36 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 84% 83% 92% 87% 87% 92% 86% 86% 92% Specificity 32% 31% 31% 34% 33% 30% 31% 31% 30% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 64% 64% 68% 68% 68% 67% 64% 64% 67% Specificity 61% 60% 56% 63% 62% 55% 58% 58% 55% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 40% 39% 32% 43% 42% 33% 36% 36% 33% Specificity 87% 86% 77% 87% 86% 78% 81% 81% 78% OR Quartile 2 2.36 2.28 5.00 3.50 3.38 4.79 2.72 2.72 4.79 p Value 0.034 0.042 0.011 0.0046 0.0059 0.014 0.024 0.024 0.014 Lower limit of 95% CI 1.07 1.03 1.44 1.47 1.42 1.38 1.14 1.14 1.38 Upper limit of 95% CI 5.24 5.06 17.4 8.32 8.02 16.7 6.47 6.47 16.7 OR Quartile 3 2.80 2.65 2.64 3.63 3.44 2.48 2.49 2.49 2.48 p Value 0.0025 0.0042 0.015 2.4E−4 4.2E−4 0.024 0.0089 0.0089 0.024 Lower limit of 95% CI 1.44 1.36 1.20 1.82 1.73 1.13 1.26 1.26 1.13 Upper limit of 95% CI 5.46 5.17 5.77 7.22 6.83 5.45 4.94 4.94 5.45 OR Quartile 4 4.36 3.85 1.65 5.11 4.51 1.74 2.31 2.31 1.74 p Value 3.2E−4 7.8E−4 0.23 7.2E−5 1.9E−4 0.19 0.028 0.028 0.19 Lower limit of 95% CI 1.96 1.75 0.727 2.28 2.05 0.764 1.09 1.09 0.764 Upper limit of 95% CI 9.71 8.44 3.75 11.4 9.96 3.96 4.90 4.90 3.96

Example 17. Use of Insulin-Like Growth Factor-Binding Protein 3 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 3 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 17.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 553 1070 630 983 624 1070 Average 1240 3690 1580 4110 1470 4670 Stdev 3000 5730 3240 6210 3070 6530 p (t-test) 0.0038 0.0019 9.6E−5 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 55 94 78 71 88 61 sCr only Median 583 1020 624 1070 622 1300 Average 1560 3540 1630 3980 1530 4640 Stdev 3740 5570 3550 6010 3310 6420 p (t-test) 0.019 0.0042 1.6E−4 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 57 92 76 73 89 60 UO only Median 672 1240 715 1350 718 1400 Average 2310 4450 2290 5070 2250 5780 Stdev 4440 6510 4340 7110 4270 7490 p (t-test) 0.029 0.0091 0.0018 Mill 20.6 172 20.6 194 20.6 194 Max 18800 18800 18800 18800 18800 18800 n (Patient) 114 34 121 27 125 23 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.64 0.66 0.64 0.64 0.68 0.68 0.69 0.70 SE 0.044 0.045 0.056 0.046 0.045 0.061 0.045 0.045 0.065 p Value 4.1E−5 0.0015 0.0037 0.0027 0.0018 0.0031 5.1E−5 2.4E−5 0.0018 nCohort Non-persistent 55 57 114 78 76 121 88 89 125 nCohort Persistent 94 92 34 71 73 27 61 60 23 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 80% 78% 85% 80% 79% 85% 85% 83% 87% Specificity 33% 30% 28% 29% 29% 27% 32% 30% 27% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 61% 59% 74% 59% 60% 78% 62% 67% 78% Specificity 67% 63% 57% 58% 59% 56% 58% 61% 55% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 35% 35% 32% 37% 37% 33% 41% 42% 39% Specificity 91% 89% 77% 85% 86% 77% 85% 85% 78% OR Quartile 2 1.92 1.53 2.26 1.70 1.58 2.16 2.70 2.18 2.49 p Value 0.090 0.27 0.12 0.17 0.24 0.18 0.020 0.061 0.16 Lower limit of 95% CI 0.902 0.720 0.805 0.796 0.741 0.693 1.17 0.963 0.695 Upper limit of 95% CI 4.09 3.25 6.36 3.64 3.35 6.71 6.23 4.92 8.92 OR Quartile 3 3.17 2.44 3.68 1.97 2.20 4.49 2.28 3.09 4.44 p Value 0.0012 0.010 0.0026 0.041 0.018 0.0026 0.016 0.0013 0.0055 Lower limit of 95% CI 1.57 1.23 1.58 1.03 1.14 1.69 1.17 1.56 1.55 Upper limit of 95% CI 6.37 4.81 8.60 3.79 4.24 11.9 4.45 6.12 12.7 OR Quartile 4 5.41 4.53 1.62 3.18 3.47 1.66 4.01 4.18 2.23 p Value 0.0011 0.0018 0.26 0.0038 0.0022 0.27 4.8E−4 3.3E−4 0.094 Lower limit of 95% CI 1.97 1.76 0.698 1.45 1.56 0.672 1.84 1.91 0.873 Upper limit of 95% CI 14.9 11.7 3.75 6.95 7.69 4.10 8.73 9.12 5.68

TABLE 17.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 547 1070 601 1070 576 1250 Average 1250 3660 1400 4110 1310 4580 Stdev 3020 5710 3000 6140 2840 6400 p (t-test) 0.0046 8.7E−4 5.5E−5 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 54 95 73 76 82 67 sCr only Median 576 983 622 1020 592 1180 Average 1570 3510 1650 3870 1530 4310 Stdev 3770 5550 3610 5920 3430 6170 p (t-test) 0.022 0.0069 6.8E−4 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 56 93 73 76 82 67 UO only Median 637 1300 642 1400 702 1440 Average 2110 4540 2110 5040 2070 5560 Stdev 4260 6370 4160 6810 4090 7080 p (t-test) 0.0079 0.0024 5.0E−4 Min 20.6 172 20.6 194 20.6 194 Max 18800 18800 18800 18800 18800 18800 n (Patient) 106 42 113 35 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.64 0.69 0.67 0.64 0.70 0.71 0.69 0.72 SE 0.044 0.045 0.051 0.044 0.045 0.054 0.043 0.044 0.056 p Value 4.1E−5 0.0015 1.8E−4 1.5E−4 0.0015 1.5E−4 5.7E−7 8.6E−6 8.5E−5 nCohort Non-persistent 54 56 106 73 73 113 82 82 117 nCohort Persistent 95 93 42 76 76 35 67 67 31 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 80% 78% 88% 82% 80% 89% 87% 85% 90% Specificity 33% 30% 30% 32% 30% 29% 34% 33% 29% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 60% 58% 74% 61% 59% 77% 64% 64% 77% Specificity 67% 62% 59% 60% 59% 58% 61% 61% 57% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 35% 34% 36% 38% 37% 37% 42% 40% 42% Specificity 91% 89% 79% 88% 86% 79% 88% 87% 79% OR Quartile 2 2.00 1.59 3.20 2.04 1.75 3.20 3.34 2.80 3.82 p Value 0.073 0.23 0.026 0.067 0.14 0.042 0.0048 0.013 0.036 Lower limit of 95% CI 0.938 0.748 1.15 0.951 0.825 1.05 1.45 1.24 1.09 Upper limit of 95% CI 4.26 3.38 8.89 4.36 3.73 9.77 7.72 6.32 13.4 OR Quartile 3 3.00 2.31 4.13 2.33 2.08 4.74 2.80 2.80 4.59 p Value 0.0021 0.016 4.3E−4 0.012 0.028 4.8E−4 0.0025 0.0025 0.0011 Lower limit of 95% CI 1.49 1.17 1.87 1.21 1.08 1.98 1.44 1.44 1.83 Upper limit of 95% CI 6.04 4.55 9.09 4.49 4.00 11.3 5.46 5.46 11.5 OR Quartile 4 5.22 4.37 2.12 4.39 3.68 2.19 5.17 4.36 2.80 p Value 0.0014 0.0023 0.061 5.4E−4 0.0017 0.061 8.7E−5 3.2E−4 0.017 Lower limit of 95% CI 1.90 1.69 0.966 1.90 1.63 0.965 2.28 1.96 1.20 Upper limit of 95% CI 14.4 11.3 4.66 10.1 8.29 4.98 11.7 9.71 6.50

TABLE 17.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 547 1070 611 1070 592 1070 Average 1250 3660 1420 4060 1360 4350 Stdev 3020 5710 3010 6120 2910 6280 p (t-test) 0.0046 0.0012 2.2E−4 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 54 95 72 77 78 71 sCr only Median 576 983 624 983 611 1070 Average 1570 3510 1680 3820 1590 4090 Stdev 3770 5550 3640 5890 3510 6060 p (L-test) 0.022 0.0090 0.0022 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 56 93 72 77 78 71 UO only Median 638 1120 679 1210 717 1300 Average 2200 4040 2160 4420 2110 4780 Stdev 4370 6090 4260 6400 4190 6630 p (t-test) 0.037 0.013 0.0046 Min 20.6 172 20.6 194 20.6 194 Max 18800 18800 18800 18800 18800 18800 n (Patient) 100 48 106 42 110 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.64 0.65 0.66 0.63 0.67 0.69 0.66 0.68 SE 0.044 0.045 0.050 0.045 0.045 0.052 0.044 0.045 0.053 p Value 4.1E−5 0.0015 0.0022 4.9E−4 0.0039 0.0012 1.6E−5 2.4E−4 9.5E−4 nCohort Non-persistent 54 56 100 72 72 106 78 78 110 nCohort Persistent 95 93 48 77 77 42 71 71 38 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 80% 78% 88% 81% 79% 88% 85% 83% 89% Specificity 33% 30% 31% 31% 29% 30% 33% 32% 30% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 60% 58% 67% 60% 58% 69% 62% 61% 68% Specificity 67% 62% 58% 60% 58% 58% 60% 59% 56% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 35% 34% 31% 38% 36% 33% 39% 38% 37% Specificity 91% 89% 78% 88% 86% 78% 87% 86% 79% OR Quartile 2 2.00 1.59 3.14 1.82 1.57 3.20 2.73 2.32 3.64 p Value 0.073 0.23 0.019 0.12 0.24 0.026 0.014 0.035 0.023 Lower limit of 95% CI 0.938 0.748 1.21 0.855 0.742 1.15 1.23 1.06 1.20 Upper limit of 95% CI 4.26 3.38 8.17 3.87 3.32 8.89 6.05 5.07 11.1 OR Quartile 3 3.00 2.31 2.76 2.20 1.97 3.02 2.47 2.21 2.80 p Value 0.0021 0.016 0.0057 0.018 0.042 0.0043 0.0072 0.018 0.0098 Lower limit of 95% CI 1.49 1.17 1.34 1.14 1.03 1.42 1.28 1.15 1.28 Upper limit of 95% CI 6.04 4.55 5.67 4.24 3.78 6.46 4.78 4.25 6.11 OR Quartile 4 5.22 4.37 1.61 4.23 3.54 1.80 4.43 3.74 2.21 p Value 0.0014 0.0023 0.23 7.3E−4 0.0023 0.14 3.6E−4 0.0012 0.054 Lower limit of 95% CI 1.90 1.69 0.744 1.83 1.57 0.818 1.96 1.68 0.988 Upper limit of 95% CI 14.4 11.3 3.49 9.77 7.99 3.98 10.0 8.30 4.93

TABLE 17.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 547 1070 611 1070 592 1070 Average 1250 3660 1420 4060 1360 4350 Stdev 3020 5710 3010 6120 2910 6280 p (t-test) 0.0046 0.0012 2.2E−4 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 54 95 72 77 78 71 sCr only Median 576 983 624 983 611 1070 Average 1570 3510 1680 3820 1590 4090 Stdev 3770 5550 3640 5890 3510 6060 p (t-test) 0.022 0.0090 0.0022 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 56 93 72 77 78 71 UO only Median 638 1070 718 1070 729 1180 Average 2240 3900 2210 4160 2160 4460 Stdev 4400 6010 4310 6260 4240 6480 p (t-test) 0.057 0.030 0.012 Min 20.6 172 20.6 194 20.6 194 Max 18800 18800 18800 18800 18800 18800 n (Patient) 98 50 103 45 107 41 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.64 0.64 0.66 0.63 0.64 0.69 0.66 0.65 SE 0.044 0.045 0.049 0.045 0.045 0.051 0.044 0.045 0.053 p Value 4.1E−5 0.0015 0.0052 4.9E−4 0.0039 0.0056 1.6E−5 2.4E−4 0.0051 nCohort Non-persistent 54 56 98 72 72 103 78 78 107 nCohort Persistent 95 93 50 77 77 45 71 71 41 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 80% 78% 86% 81% 79% 87% 85% 83% 88% Specificity 33% 30% 31% 31% 29% 30% 33% 32% 30% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 60% 58% 64% 60% 58% 64% 62% 61% 63% Specificity 67% 62% 57% 60% 58% 56% 60% 59% 55% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 35% 34% 30% 38% 36% 31% 39% 38% 34% Specificity 91% 89% 78% 88% 86% 78% 87% 86% 79% OR Quartile 2 2.00 1.59 2.71 1.82 1.57 2.80 2.73 2.32 3.07 p Value 0.073 0.23 0.031 0.12 0.24 0.035 0.014 0.035 0.032 Lower limit of 95% CI 0.938 0.748 1.09 0.855 0.742 1.07 1.23 1.06 1.10 Upper limit of 95% CI 4.26 3.38 6.71 3.87 3.32 7.29 6.05 5.07 8.54 OR Quartile 3 3.00 2.31 2.37 2.20 1.97 2.34 2.47 2.21 2.13 p Value 0.0021 0.016 0.016 0.018 0.042 0.022 0.0072 0.018 0.045 Lower limit of 95% CI 1.49 1.17 1.17 1.14 1.03 1.13 1.28 1.15 1.02 Upper limit of 95% CI 6.04 4.55 4.79 4.24 3.78 4.82 4.78 4.25 4.47 OR Quartile 4 5.22 4.37 1.48 4.23 3.54 1.57 4.43 3.74 1.89 p Value 0.0014 0.0023 0.32 7.3E−4 0.0023 0.26 3.6E−4 0.0012 0.11 Lower limit of 95% CI 1.90 1.69 0.686 1.83 1.57 0.718 1.96 1.68 0.857 Upper limit of 95% CI 14.4 11.3 3.19 9.77 7.99 3.44 10.0 8.30 4.19

TABLE 17.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RTFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 547 1070 601 1070 592 1070 Average 1250 3600 1280 4080 1240 4310 Stdev 3080 5660 2760 6100 2670 6230 p (t-test) 0.0060 5.8E−4 1.5E−4 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 16600 18800 16600 18800 16600 18800 n (Patient) 52 97 69 80 74 75 sCr only Median 569 1020 611 1070 592 1070 Average 1560 3500 1510 3910 1440 4180 Stdev 3810 5520 3440 5900 3320 6060 p (t-test) 0.023 0.0034 7.7E−4 Min 30.5 20.6 21.1 20.6 21.1 20.6 Max 18800 18800 18800 18800 18800 18800 n (Patient) 55 94 70 79 76 73 UO only Median 630 1070 642 1070 680 1200 Average 2140 4010 2140 4120 2100 4340 Stdev 4320 6020 4270 6170 4210 6310 p (t-test) 0.031 0.024 0.012 Min 20.6 172 20.6 172 20.6 172 Max 18800 18800 18800 18800 18800 18800 n (Patient) 96 52 99 49 102 46 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.65 0.65 0.66 0.65 0.64 0.68 0.68 0.65 SE 0.044 0.045 0.048 0.044 0.045 0.050 0.044 0.044 0.050 p Value 5.2E−5 6.9E−4 0.0018 3.1E−4 8.1E−4 0.0040 3.8E−5 3.2E−5 0.0023 nCohort Non-persistent 52 55 96 69 70 99 74 76 102 nCohort Persistent 97 94 52 80 79 49 75 73 46 Cutoff Quartile 2 401 401 410 401 401 410 401 401 410 Sensitivity 79% 79% 87% 80% 80% 86% 83% 84% 87% Specificity 33% 31% 31% 30% 30% 30% 32% 33% 30% Cutoff Quartile 3 794 794 801 794 794 801 794 794 801 Sensitivity 60% 59% 65% 60% 59% 65% 61% 62% 65% Specificity 67% 64% 58% 61% 60% 58% 61% 61% 57% Cutoff Quartile 4 1830 1830 1840 1830 1830 1840 1830 1830 1840 Sensitivity 35% 35% 31% 38% 38% 31% 39% 40% 33% Specificity 92% 91% 78% 88% 89% 78% 88% 88% 78% OR Quartile 2 1.87 1.66 2.92 1.75 1.69 2.61 2.29 2.49 2.91 p Value 0.11 0.19 0.020 0.14 0.17 0.038 0.035 0.022 0.029 Lower limit of 95% CI 0.874 0.777 1.18 0.826 0.797 1.05 1.06 1.14 1.12 Upper limit of 95% CI 4.00 3.52 7.23 3.71 3.57 6.47 4.95 5.45 7.57 OR Quartile 3 3.06 2.47 2.64 2.33 2.20 2.55 2.46 2.46 2.47 p Value 0.0019 0.0098 0.0065 0.012 0.018 0.0097 0.0074 0.0073 0.014 Lower limit of 95% CI 1.51 1.24 1.31 1.21 1.14 1.26 1.27 1.28 1.20 Upper limit of 95% CI 6.21 4.90 5.33 4.51 4.25 5.20 4.76 4.76 5.09 OR Quartile 4 6.48 5.41 1.59 4.58 4.74 1.54 4.55 4.91 1.76 p Value 8.9E−4 0.0011 0.23 5.7E−4 4.2E−4 0.27 3.9E−4 2.0E−4 0.15 Lower limit of 95% CI 2.15 1.97 0.741 1.93 2.00 0.715 1.97 2.12 0.810 Upper limit of 95% CI 19.5 14.9 3.40 10.9 11.3 3.34 10.5 11.4 3.82

Example 18. Use of Insulin-Like Growth Factor-Binding Protein 4 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 4 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 18.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0669 1.14 0.0851 1.75 0.0895 1.36 Average 4.30 14.0 4.43 15.5 5.82 13.2 Stdev 16.9 21.1 16.5 22.2 18.3 20.0 p (t-test) 0.0028 0.0011 0.040 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 61.5 n (Patient) 98 51 106 43 113 36 sCr only Median 0.0851 1.13 0.0851 1.75 0.118 1.36 Average 4.35 13.7 4.36 15.6 5.81 12.9 Stdev 17.0 21.0 16.5 22.1 18.5 19.6 p (L-Lest) 0.0037 8.2E−4 0.046 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 61.5 n (Patient) 97 52 106 43 111 38 UO only Median 0.146 1.03 0.126 1.14 0.126 1.14 Average 6.99 12.6 6.94 13.3 6.94 13.3 Stdev 19.0 18.8 18.9 19.1 18.9 19.1 p (t-test) 0.24 0.20 0.20 Min 0.000212 0.000339 0.000212 0.000339 0.000212 0.000339 Max 120 50.9 120 50.9 120 50.9 n (Patient) 130 18 131 17 131 17 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.68 0.67 0.66 0.69 0.68 0.67 0.65 0.63 0.67 SE 0.048 0.048 0.074 0.050 0.051 0.075 0.055 0.054 0.075 p Value 1.9E−4 5.2E−4 0.033 1.4E−4 3.0E−4 0.027 0.0063 0.013 0.027 nCohort Non-persistent 98 97 130 106 106 131 113 111 131 nCohort Persistent 51 52 18 43 43 17 36 38 17 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 88% 87% 100% 86% 84% 100% 86% 82% 100% Specificity 22% 22% 21% 21% 20% 21% 20% 19% 21% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 63% 62% 61% 67% 67% 65% 64% 63% 65% Specificity 56% 56% 52% 57% 57% 52% 54% 54% 52% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 43% 42% 39% 47% 47% 41% 42% 42% 41% Specificity 84% 84% 77% 83% 83% 77% 80% 80% 77% OR Quartile 2 2.17 1.78 9.83 1.62 1.27 9.21 1.58 1.03 9.21 p Value 0.12 0.23 0.11 0.34 0.62 0.13 0.39 0.95 0.13 Lower limit of 95% CI 0.819 0.700 0.574 0.605 0.496 0.537 0.555 0.401 0.537 Upper limit of 95% CI 5.76 4.51 168 4.31 3.25 158 4.53 2.67 158 OR Quartile 3 2.15 2.01 1.67 2.70 2.70 1.98 2.08 2.02 1.98 p Value 0.030 0.047 0.32 0.0089 0.0089 0.20 0.065 0.070 0.20 Lower limit of 95% CI 1.08 1.01 0.610 1.28 1.28 0.691 0.957 0.946 0.691 Upper limit of 95% CI 4.31 4.00 4.58 5.69 5.69 5.67 4.50 4.30 5.67 OR Quartile 4 3.89 3.71 2.12 4.25 4.25 2.36 2.80 2.94 2.36 p Value 5.5E−4 8.2E−4 0.15 3.0E−4 3.0E−4 0.11 0.012 0.0078 0.11 Lower limit of 95% CI 1.80 1.72 0.756 1.94 1.94 0.826 1.25 1.33 0.826 Upper limit of 95% CI 8.40 8.00 5.95 9.32 9.32 6.72 6.25 6.52 6.72

TABLE 18.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0328 1.17 0.0328 1.58 0.0851 1.17 Average 4.50 12.5 4.55 13.5 6.01 11.4 Stdev 17.5 20.2 17.2 20.9 18.9 18.6 p (t-test) 0.011 0.0058 0.11 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 61.5 n (Patient) 91 58 98 51 105 44 sCr only Median 0.0487 1.13 0.0328 1.67 0.0851 1.13 Average 4.51 12.5 4.57 13.3 6.10 11.0 Stdev 17.5 20.2 17.2 20.7 19.1 18.3 p (t-test) 0.012 0.0070 0.15 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 61.5 n (Patient) 91 58 97 52 103 46 UO only Median 0.126 1.13 0.126 1.13 0.126 1.13 Average 7.28 9.57 7.22 9.96 7.22 9.96 Stdev 19.5 16.6 19.4 16.9 19.4 16.9 p (t-test) 0.58 0.52 0.52 Min 0.000212 0.000339 0.000212 0.000339 0.000212 0.000339 Max 120 50.9 120 50.9 120 50.9 n (Patient) 123 25 124 24 124 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.67 0.65 0.70 0.69 0.65 0.66 0.64 0.65 SE 0.046 0.046 0.064 0.047 0.047 0.065 0.051 0.051 0.065 p Value 4.2E−5 2.0E−4 0.021 1.6E−5 4.1E−5 0.018 0.0012 0.0049 0.018 nCohort Non-persistent 91 91 123 98 97 124 105 103 124 nCohort Persistent 58 58 25 51 52 24 44 46 24 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 90% 88% 100% 88% 87% 100% 89% 85% 100% Specificity 24% 23% 22% 22% 22% 22% 22% 20% 22% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 62% 60% 60% 67% 65% 62% 64% 61% 62% Specificity 57% 56% 52% 58% 58% 52% 55% 54% 52% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 41% 41% 36% 43% 44% 38% 41% 41% 38% Specificity 85% 85% 77% 84% 85% 77% 81% 82% 77% OR Quartile 2 2.76 2.19 14.5 2.17 1.78 13.8 2.19 1.43 13.8 p Value 0.040 0.099 0.064 0.12 0.23 0.069 0.14 0.46 0.069 Lower limit of 95% CI 1.05 0.864 0.857 0.819 0.700 0.814 0.774 0.559 0.814 Upper limit of 95% CI 7.30 5.53 246 5.76 4.51 235 6.19 3.64 235 OR Quartile 3 2.18 1.94 1.63 2.78 2.58 1.84 2.16 1.85 1.84 p Value 0.023 0.052 0.28 0.0046 0.0079 0.18 0.037 0.088 0.18 Lower limit of 95% CI 1.11 0.993 0.678 1.37 1.28 0.748 1.05 0.913 0.748 Upper limit of 95% CI 4.28 3.79 3.90 5.64 5.19 4.51 4.46 3.76 4.51 OR Quartile 4 3.88 3.88 1.91 3.89 4.34 2.06 2.94 3.11 2.06 p Value 5.8E−4 5.8E−4 0.17 5.5E−4 2.1E−4 0.13 0.0063 0.0038 0.13 Lower limit of 95% CI 1.79 1.79 0.761 1.80 2.00 0.814 1.36 1.44 0.814 Upper limit of 95% CI 8.41 8.41 4.78 8.40 9.42 5.20 6.38 6.72 5.20

TABLE 18.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0328 1.21 0.0328 1.46 0.0407 1.21 Average 4.49 11.9 4.29 12.7 5.99 10.7 Stdev 17.9 19.6 17.5 20.0 19.3 17.9 p (t-test) 0.018 0.0077 0.15 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 61.5 n (Patient) 86 63 90 59 98 51 sCr only Median 0.0328 1.17 0.0328 1.46 0.0851 1.13 Average 4.55 11.7 4.31 12.7 6.06 10.5 Stdev 18.0 19.5 17.5 20.0 19.4 17.8 p (t-test) 0.022 0.0080 0.17 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 61.5 n (Patient) 85 64 90 59 97 52 UO only Median 0.122 1.13 0.126 1.14 0.126 1.14 Average 7.21 9.31 7.15 9.61 7.15 9.61 Stdev 19.8 15.9 19.7 16.1 19.7 16.1 p (t-test) 0.58 0.52 0.52 Mill 0.000212 0.000312 0.000212 0.000312 0.000212 0.000312 Max 120 50.9 120 50.9 120 50.9 n (Patient) 116 32 117 31 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.69 0.68 0.64 0.71 0.70 0.65 0.66 0.64 0.65 SE 0.045 0.045 0.058 0.044 0.045 0.059 0.048 0.049 0.059 p Value 3.0E−5 9.1E−5 0.014 1.7E−6 1.2E−5 0.012 6.5E−4 0.0048 0.012 nCohort Non-persistent 86 85 116 90 90 117 98 97 117 nCohort Persistent 63 64 32 59 59 31 51 52 31 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 89% 88% 97% 88% 86% 97% 88% 85% 97% Specificity 24% 24% 22% 23% 22% 22% 22% 21% 22% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 62% 61% 59% 66% 64% 61% 63% 60% 61% Specificity 58% 58% 53% 60% 59% 53% 56% 55% 53% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 40% 39% 34% 42% 42% 35% 39% 38% 35% Specificity 85% 85% 78% 86% 86% 78% 82% 81% 78% OR Quartile 2 2.58 2.15 8.96 2.26 1.82 8.57 2.17 1.43 8.57 p Value 0.045 0.093 0.035 0.085 0.19 0.039 0.12 0.44 0.039 Lower limit of 95% CI 1.02 0.881 1.17 0.894 0.744 1.12 0.819 0.581 1.12 Upper limit of 95% CI 6.53 5.27 68.8 5.72 4.46 65.9 5.76 3.51 65.9 OR Quartile 3 2.26 2.12 1.62 2.92 2.59 1.78 2.15 1.78 1.78 p Value 0.016 0.026 0.23 0.0021 0.0059 0.16 0.030 0.099 0.16 Lower limit of 95% CI 1.16 1.10 0.733 1.48 1.32 0.795 1.08 0.898 0.795 Upper limit of 95% CI 4.39 4.11 3.59 5.80 5.11 4.01 4.31 3.52 4.01 OR Quartile 4 3.69 3.55 1.81 4.36 4.36 1.92 2.87 2.74 1.92 p Value 9.7E−4 0.0014 0.17 2.3E−4 2.3E−4 0.13 0.0066 0.0091 0.13 Lower limit of 95% CI 1.70 1.64 0.775 1.99 1.99 0.819 1.34 1.29 0.819 Upper limit of 95% CI 8.03 7.71 4.24 9.52 9.52 4.53 6.13 5.85 4.53

TABLE 18.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0328 1.28 0.0328 1.46 0.0407 1.21 Average 4.40 11.9 4.29 12.7 5.35 11.7 Stdev 18.0 19.4 17.5 20.0 18.2 19.6 p (t-test) 0.016 0.0077 0.049 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 73.0 n (Patient) 85 64 90 59 96 53 sCr only Median 0.0328 1.17 0.0328 1.40 0.0487 1.17 Average 4.55 11.7 4.34 12.5 5.41 11.5 Stdev 18.0 19.5 17.6 19.9 18.3 19.5 p (t-test) 0.022 0,0097 0.059 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 73.0 120 73.0 120 73.0 n (Patient) 85 64 89 60 95 54 UO only Median 0.0873 1.13 0.104 1.13 0.104 1.13 Average 6.98 9.79 6.97 9.98 6.97 9.98 Stdev 19.9 16.1 19.7 16.4 19.7 16.4 p (t-test) 0.44 0.42 0.42 Min 0.000212 0.000312 0.000212 0.000312 0.000212 0.000312 Max 120 50.9 120 50.9 120 50.9 n (Patient) 112 36 114 34 114 34 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.68 0.66 0.71 0.70 0.66 0.67 0.65 0.66 SE 0.044 0.045 0.055 0.044 0.045 0.056 0.048 0.048 0.056 p Value 9.7E−6 9.1E−5 0.0029 1.7E−6 6.6E−6 0.0049 4.8E−4 0.0012 0.0049 nCohort Non-persistent 85 85 112 90 89 114 96 95 114 nCohort Persistent 64 64 36 59 60 34 53 54 34 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 89% 88% 97% 88% 87% 97% 87% 85% 97% Specificity 25% 24% 23% 23% 22% 23% 22% 21% 23% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 62% 61% 61% 66% 65% 62% 62% 61% 62% Specificity 59% 58% 54% 60% 60% 54% 56% 56% 54% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 41% 39% 36% 42% 42% 35% 40% 39% 35% Specificity 86% 85% 79% 86% 85% 78% 82% 82% 78% OR Quartile 2 2.67 2.15 10.6 2.26 1.88 9.75 1.84 1.53 9.75 p Value 0.038 0.093 0.023 0.085 0.17 0.028 0.20 0.35 0.028 Lower limit of 95% CI 1.06 0.881 1.38 0.894 0.769 1.27 0.725 0.624 1.27 Upper limit of 95% CI 6.75 5.27 81.0 5.72 4.61 74.8 4.67 3.77 74.8 OR Quartile 3 2.38 2.12 1.81 2.92 2.73 1.86 2.12 1.98 1.86 p Value 0.011 0.026 0.13 0.0021 0.0037 0.12 0.032 0.049 0.12 Lower limit of 95% CI 1.22 1.10 0.843 1.48 1.39 0.849 1.07 1.00 0.849 Upper limit of 95% CI 4.63 4.11 3.90 5.80 5.39 4.07 4.21 3.92 4.07 OR Quartile 4 4.16 3.55 2.07 4.36 4.18 1.94 3.05 2.92 1.94 p Value 3.9E−4 0.0014 0.080 2.3E−4 3.3E−4 0.12 0.0040 0.0056 0.12 Lower limit of 95% CI 1.89 1.64 0.916 1.99 1.91 0.845 1.43 1.37 0.845 Upper limit of 95% CI 9.16 7.71 4.69 9.52 9.12 4.46 6.52 6.23 4.46

TABLE 18.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.0328 1.35 0.0169 1.58 0.0328 1.28 Average 2.93 13.3 2.79 14.2 5.25 11.5 Stdev 12.9 23.2 12.6 23.7 18.5 19.2 p (t-test) 7.0E−4 2.2E−4 0.049 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 120 73.0 n (Patient) 82 67 86 63 93 56 sCr only Median 0.0328 1.28 0.0328 1.52 0.0328 1.28 Average 3.05 13.4 2.91 14.2 4.09 13.5 Stdev 12.9 23.4 12.6 23.9 14.1 24.0 p (t-test) 8.1E−4 2.5E−4 0.0031 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 83 66 87 62 93 56 UO only Median 0.0851 1.14 0.0873 1.17 0.0873 1.17 Average 6.64 10.7 6.58 11.0 6.58 11.0 Stdev 19.6 16.9 19.5 17.0 19.5 17.0 p (t-test) 0.26 0.22 0.22 Min 0.000212 0.000312 0.000212 0.000312 0.000212 0.000312 Max 120 50.9 120 50.9 120 50.9 n (Patient) 111 37 112 36 112 36 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.70 0.68 0.73 0.72 0.68 0.67 0.68 0.68 SE 0.043 0.044 0.054 0.043 0.043 0.054 0.047 0.047 0.054 p Value 1.6E−6 5.9E−6 0.0011 5.4E−8 2.5E−7 8.7E−4 2.2E−4 1.4E−4 8.7E−4 nCohort Non-persistent 82 83 111 86 87 112 93 93 112 nCohort Persistent 67 66 37 63 62 36 56 56 36 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 88% 88% 97% 87% 87% 97% 86% 86% 97% Specificity 24% 24% 23% 23% 23% 23% 22% 22% 23% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 63% 62% 62% 67% 66% 64% 62% 62% 64% Specificity 60% 59% 54% 62% 61% 54% 57% 57% 54% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 42% 41% 38% 44% 44% 39% 41% 41% 39% Specificity 88% 87% 79% 88% 87% 79% 84% 84% 79% OR Quartile 2 2.38 2.30 11.0 2.08 2.01 10.6 1.64 1.64 10.6 p Value 0.057 0.068 0.021 0.11 0.12 0.023 0.28 0.28 0.023 Lower limit of 95% CI 0.973 0.941 1.44 0.852 0.823 1.38 0.670 0.670 1.38 Upper limit of 95% CI 5.82 5.63 84.3 5.10 4.93 81.0 4.03 4.03 81.0 OR Quartile 3 2.49 2.36 1.93 3.21 3.04 2.12 2.21 2.21 2.12 p Value 0.0069 0.011 0.090 7.8E−4 0.0013 0.058 0.022 0.022 0.058 Lower limit of 95% CI 1.29 1.22 0.902 1.63 1.54 0.975 1.12 1.12 0.975 Upper limit of 95% CI 4.84 4.58 4.14 6.34 6.01 4.59 4.36 4.36 4.59 OR Quartile 4 5.17 4.53 2.33 6.08 5.33 2.46 3.62 3.62 2.46 p Value 8.7E−5 2.2E−4 0.040 1.8E−5 4.9E−5 0.030 0.0010 0.0010 0.030 Lower limit of 95% CI 2.28 2.03 1.04 2.66 2.38 1.09 1.68 1.68 1.09 Upper limit of 95% CI 11.7 10.1 5.22 13.9 11.9 5.55 7.81 7.81 5.55

Example 19. Use of Insulin-Like Growth Factor-Binding Protein 4 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 4 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 19.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000473 0.899 0.0669 0.866 0.0328 1.13 Average 2.51 10.6 3.39 12.3 3.11 14.1 Stdev 14.9 20.4 13.3 22.8 12.6 24.1 p (t-test) 0.011 0.0039 4.0E−4 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 55 94 78 71 88 61 sCr only Median 0.000473 0.899 0.0407 0.872 0.0328 1.40 Average 2.65 10.7 3.38 12.0 2.97 14.5 Stdev 14.7 20.6 13.5 22.6 12.5 24.2 p (t-test) 0.011 0.0049 2.0E−4 Mill 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 57 92 76 73 89 60 UO only Median 0.146 0.869 0.126 0.927 0.126 1.35 Average 7.17 9.31 6.99 10.7 6.77 12.5 Stdev 19.9 15.6 19.4 16.9 19.1 17.7 p (t-test) 0.57 0.36 0.18 Min 0.000212 0.000286 0.000212 0.000286 0.000212 0.000301 Max 120 50.9 120 50.9 120 50.9 n (Patient) 114 34 121 27 125 23 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.73 0.59 0.64 0.65 0.62 0.68 0.69 0.67 SE 0.040 0.041 0.057 0.045 0.045 0.062 0.046 0.045 0.066 p Value 5.1E−9 2.8E−8 0.12 0.0022 0.0012 0.055 1.1E−4 2.4E−5 0.0084 nCohort Non-persistent 55 57 114 78 76 121 88 89 125 nCohort Persistent 94 92 34 71 73 27 61 60 23 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 86% 86% 85% 85% 84% 89% 85% 83% 96% Specificity 27% 26% 19% 22% 21% 20% 22% 20% 21% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 63% 63% 56% 58% 59% 59% 62% 67% 65% Specificity 71% 70% 52% 56% 58% 52% 58% 61% 53% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 38% 38% 35% 37% 37% 37% 39% 43% 43% Specificity 96% 95% 78% 85% 86% 78% 84% 87% 78% OR Quartile 2 2.34 2.17 1.39 1.52 1.36 1.98 1.59 1.27 5.78 p Value 0.046 0.068 0.54 0.33 0.47 0.30 0.30 0.59 0.094 Lower limit of 95% CI 1.02 0.945 0.482 0.658 0.592 0.550 0.666 0.540 0.744 Upper limit of 95% CI 5.38 4.99 3.99 3.51 3.11 7.12 3.80 2.98 44.9 OR Quartile 3 4.11 4.01 1.36 1.77 1.97 1.58 2.28 3.09 2.10 p Value 1.1E−4 1.2E−4 0.44 0.085 0.041 0.29 0.016 0.0013 0.12 Lower limit of 95% CI 2.01 1.98 0.629 0.924 1.03 0.678 1.17 1.56 0.830 Upper limit of 95% CI 8.41 8.15 2.94 3.39 3.78 3.68 4.45 6.12 5.30 OR Quartile 4 16.4 11.1 1.94 3.18 3.47 2.05 3.43 4.91 2.79 p Value 1.9E−4 1.4E−4 0.12 0.0038 0.0022 0.11 0.0017 8.7E−5 0.030 Lower limit of 95% CI 3.78 3.21 0.845 1.45 1.56 0.840 1.59 2.22 1.10 Upper limit of 95% CI 71.7 38.1 4.46 6.95 7.69 4.99 7.39 10.9 7.06

TABLE 19.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000472 0.927 0.0328 1.03 0.000747 1.35 Average 2.53 10.5 3.29 11.8 2.98 13.3 Stdev 15.0 20.3 13.7 22.2 13.0 23.2 p (t-test) 0.013 0.0059 8.0E−4 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 54 95 73 76 82 67 sCr only Median 0.000473 0.927 0.0328 0.899 0.0169 1.35 Average 2.66 10.6 3.46 11.6 3.09 13.1 Stdev 14.8 20.5 13.8 22.2 13.0 23.2 p (t-test) 0.013 0.0082 0.0011 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 56 93 73 76 82 67 UO only Median 0.118 1.03 0.118 1.14 0.118 1.35 Average 7.08 9.15 6.89 10.2 6.66 11.5 Stdev 20.4 15.1 19.8 16.1 19.5 16.7 p (t-test) 0.55 0.37 0.21 Min 0.000212 0.000286 0.000212 0.000286 0.000212 0.000301 Max 120 50.9 120 50.9 120 50.9 n (Patient ) 106 42 113 35 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.73 0.63 0.67 0.65 0.65 0.71 0.70 0.70 SE 0.040 0.040 0.053 0.044 0.045 0.056 0.043 0.043 0.057 p Value 1.0E−9 6.8E−9 0.017 8.6E−5 6.0E−4 0.0059 6.9E−7 3.3E−6 5.7E−4 nCohort Non-persistent 54 56 106 73 73 113 82 82 117 nCohort Persistent 95 93 42 76 76 35 67 67 31 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 86% 86% 88% 86% 84% 91% 87% 85% 97% Specificity 28% 27% 21% 23% 22% 21% 23% 22% 22% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 63% 63% 60% 61% 59% 63% 66% 66% 68% Specificity 72% 71% 54% 60% 59% 54% 62% 62% 55% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 38% 38% 38% 37% 36% 40% 40% 40% 45% Specificity 96% 95% 80% 86% 85% 80% 87% 87% 80% OR Quartile 2 2.43 2.25 1.94 1.79 1.50 2.88 1.94 1.60 8.57 p Value 0.037 0.056 0.21 0.17 0.34 0.10 0.13 0.28 0.039 Lower limit of 95% CI 1.05 0.979 0.681 0.776 0.653 0.811 0.815 0.684 1.12 Upper limit of 95% CI 5.59 5.18 5.51 4.15 3.43 10.2 4.64 3.76 65.9 OR Quartile 3 4.46 4.34 1.71 2.33 2.08 1.99 3.15 3.15 2.54 p Value 5.6E−5 6.0E−5 0.15 0.012 0.028 0.085 8.5E−4 8.5E−4 0.029 Lower limit of 95% CI 2.15 2.12 0.829 1.21 1.08 0.911 1.60 1.60 1.10 Upper limit of 95% CI 9.22 8.89 3.53 4.49 4.00 4.33 6.17 6.17 5.85 OR Quartile 4 15.9 10.7 2.49 3.68 3.11 2.61 4.36 4.36 3.37 p Value 2.3E−4 1.8E−4 0.023 0.0017 0.0052 0.021 3.2E−4 3.2E−4 0.0047 Lower limit of 95% CI 3.64 3.10 1.14 1.63 1.40 1.15 1.96 1.96 1.45 Upper limit of 95% CI 69.1 36.7 5.46 8.29 6.88 5.90 9.71 9.71 7.81

TABLE 19.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000472 0.927 0.0328 0.927 0.000747 1.21 Average 2.53 10.5 3.32 11.6 3.07 12.6 Stdev 15.0 20.3 13.8 22.0 13.3 22.7 p (t-test) 0.013 0.0070 0.0019 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 54 95 72 77 78 71 sCr only Median 0.000473 0.927 0.0328 0.872 0.0169 1.14 Average 2.66 10.6 3.50 11.5 3.24 12.4 Stdev 14.8 20.5 13.8 22.1 13.3 22.8 p (t-test) 0.013 0.0098 0.0028 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 56 93 72 77 78 71 UO only Median 0.122 0.899 0.104 1.13 0.104 1.28 Average 7.45 8.12 7.12 9.05 6.87 9.97 Stdev 20.9 14.4 20.4 15.1 20.0 15.6 p (t-test) 0.84 0.58 0.39 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 50.9 120 50.9 120 50.9 n (Patient) 100 48 106 42 110 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.73 0.59 0.68 0.66 0.63 0.71 0.69 0.66 SE 0.040 0.040 0.051 0.044 0.044 0.053 0.043 0.044 0.054 p Value 1.0E−9 6.8E−9 0.062 5.7E−5 4.2E−4 0.017 1.5E−6 1.9E−5 0.0031 nCohort Non-persistent 54 56 100 72 72 106 78 78 110 nCohort Persistent 95 93 48 77 77 42 71 71 38 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 86% 86% 85% 86% 84% 88% 86% 85% 92% Specificity 28% 27% 20% 24% 22% 21% 23% 22% 22% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 63% 63% 56% 61% 60% 60% 65% 63% 63% Specificity 72% 71% 53% 61% 60% 54% 63% 62% 55% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 38% 38% 35% 36% 35% 38% 39% 38% 42% Specificity 96% 95% 80% 86% 85% 80% 87% 86% 81% OR Quartile 2 2.43 2.25 1.46 1.85 1.55 1.94 1.83 1.52 3.26 p Value 0.037 0.056 0.43 0.15 0.30 0.21 0.16 0.33 0.067 Lower limit of 95% CI 1.05 0.979 0.572 0.802 0.675 0.681 0.781 0.658 0.921 Upper limit of 95% CI 5.59 5.18 3.75 4.29 3.55 5.51 4.29 3.51 11.5 OR Quartile 3 4.46 4.34 1.45 2.46 2.20 1.71 3.11 2.77 2.06 p Value 5.6E−5 6.0E−5 0.29 0.0074 0.018 0.15 9.0E−4 0.0027 0.062 Lower limit of 95% CI 2.15 2.12 0.725 1.27 1.14 0.829 1.59 1.43 0.963 Upper limit of 95% CI 9.22 8.89 2.90 4.76 4.24 3.53 6.07 5.38 4.39 OR Quartile 4 15.9 10.7 2.19 3.54 2.99 2.49 4.43 3.74 3.08 p Value 2.3E−4 1.8E−4 0.045 0.0023 0.0068 0.023 3.6E−4 0.0012 0.0058 Lower limit of 95% CI 3.64 3.10 1.02 1.57 1.35 1.14 1.96 1.68 1.38 Upper limit of 95% CI 69.1 36.7 4.73 7.99 6.63 5.46 10.0 8.30 6.86

TABLE 19.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000472 0.927 0.0328 0.927 0.000747 1.21 Average 2.53 10.5 3.32 11.6 3.07 12.6 Stdev 15.0 20.3 13.8 22.0 13.3 22.7 p (t-test) 0.013 0.0070 0.0019 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 54 95 72 77 78 71 sCr only Median 0.000473 0.927 0.0328 0.872 0.0169 1.14 Average 2.66 10.6 3.50 11.5 3.24 12.4 Stdev 14.8 20.5 13.8 22.1 13.3 22.8 p (t-test) 0.013 0.0098 0.0028 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 56 93 72 77 78 71 UO only Median 0.142 0.869 0.0895 0.927 0.0895 1.14 Average 7.60 7.79 7.32 8.47 7.05 9.27 Stdev 21.1 14.2 20.6 14.7 20.3 15.2 p (t-test) 0.95 0.74 0.53 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 50.9 120 50.9 120 50.9 n (Patient) 98 50 103 45 107 41 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.73 0.58 0.68 0.66 0.61 0.71 0.69 0.64 SE 0.040 0.040 0.050 0.044 0.044 0.052 0.043 0.044 0.053 p Value 1.0E−9 6.8E−9 0.10 5.7E−5 4.2E−4 0.028 1.5E−6 1.9E−5 0.0063 nCohort Non-persistnet 54 56 98 72 72 103 78 78 107 nCohort Persistent 95 93 50 77 77 45 71 71 41 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 86% 86% 84% 86% 84% 87% 86% 85% 90% Specificity 28% 27% 19% 24% 22% 20% 23% 22% 21% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 63% 63% 54% 61% 60% 58% 65% 63% 61% Specificity 72% 71% 52% 61% 60% 53% 63% 62% 54% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 38% 38% 34% 36% 35% 36% 39% 38% 39% Specificity 96% 95% 80% 86% 85% 80% 87% 86% 80% OR Quartile 2 2.43 2.25 1.26 1.85 1.55 1.66 1.83 1.52 2.53 p Value 0.037 0.056 0.61 0.15 0.30 0.31 0.16 0.33 0.11 Lower limit of 95% CI 1.05 0.979 0.510 0.802 0.675 0.622 0.781 0.658 0.818 Upper limit of 95% CI 5.59 5.18 3.13 4.29 3.55 4.45 4.29 3.51 7.84 OR Quartile 3 4.46 4.34 1.27 2.46 2.20 1.57 3.11 2.77 1.85 p Value 5.6E−5 6.0E−5 0.49 0.0074 0.018 0.21 9.0E−4 0.0027 0.10 Lower limit of 95% CI 2.15 2.12 0.643 1.27 1.14 0.773 1.59 1.43 0.888 Upper limit of 95% CI 9.22 8.89 2.52 4.76 4.24 3.18 6.07 5.38 3.85 OR Quartile 4 15.9 10.7 2.01 3.54 2.99 2.15 4.43 3.74 2.62 p Value 2.3E−4 1.8E−4 0.073 0.0023 0.0068 0.053 3.6E−4 0.0012 0.017 Lower limit of 95% CI 3.64 3.10 0.936 1.57 1.35 0.991 1.96 1.68 1.19 Upper limit of 95% CI 69.1 36.7 4.31 7.99 6.63 4.68 10.0 8.30 5.77

TABLE 19.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 0.000472 0.927 0.0328 0.899 0.0169 1.14 Average 2.38 10.4 3.27 11.4 3.06 12.1 Stdev 15.2 20.2 14.0 21.7 13.6 22.2 p (t-test) 0.013 0.0089 0.0032 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 52 97 69 80 74 75 sCr only Median 0.000473 1.03 0.0328 0.927 0.000747 1.21 Average 2.48 10.6 3.22 11.5 2.98 12.4 Stdev 14.9 20.4 13.9 21.8 13.4 22.4 p (t-test) 0.011 0.0073 0.0020 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 110 120 110 120 110 120 n (Patient) 55 94 70 79 76 73 UO only Median 0.104 0.899 0.0895 0.927 0.0873 1.13 Average 7.15 8.61 7.02 8.96 6.82 9.54 Stdev 21.0 14.8 20.7 15.2 20.4 15.5 p (t-test) 0.66 0.56 0.42 Min 0.000212 0.000212 0.000212 0.000212 0.000212 0.000212 Max 120 50.9 120 50.9 120 50.9 n (Patient) 96 52 99 49 102 46 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.75 0.60 0.67 0.68 0.62 0.70 0.71 0.65 SE 0.040 0.040 0.050 0.044 0.044 0.050 0.043 0.043 0.050 p Value 4.5E−10 5.2E−10 0.036 7.9E−5 5.1E−5 0.015 4.9E−6 1.4E−6 0.0026 nCohort Non-persistnet 52 55 96 69 70 99 74 76 102 nCohort Persistent 97 94 52 80 79 49 75 73 46 Cutoff Quartile 2 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 0.000339 Sensitivity 86% 86% 85% 85% 85% 88% 85% 85% 91% Specificity 27% 27% 20% 23% 23% 21% 23% 22% 23% Cutoff Quartile 3 0.170 0.170 0.205 0.170 0.170 0.205 0.170 0.170 0.205 Sensitivity 63% 64% 56% 60% 61% 57% 63% 64% 61% Specificity 73% 73% 53% 61% 61% 54% 62% 63% 55% Cutoff Quartile 4 2.99 2.99 3.01 2.99 2.99 3.01 2.99 2.99 3.01 Sensitivity 38% 38% 37% 36% 37% 37% 39% 40% 39% Specificity 98% 96% 81% 87% 87% 81% 88% 88% 81% OR Quartile 2 2.18 2.34 1.36 1.71 1.65 1.93 1.74 1.62 3.06 p Value 0.066 0.046 0.51 0.20 0.23 0.19 0.20 0.26 0.052 Lower limit of 95% CI 0.948 1.02 0.549 0.746 0.721 0.724 0.750 0.702 0.992 Upper limit of 95% CI 5.03 5.38 3.36 3.92 3.79 5.14 4.01 3.75 9.42 OR Quartile 3 4.60 4.71 1.43 2.33 2.47 1.54 2.76 3.10 1.89 p Value 5.1E−5 3.0E−5 0.30 0.012 0.0074 0.22 0.0027 9.1E−4 0.078 Lower limit of 95% CI 2.20 2.27 0.725 1.21 1.27 0.771 1.42 1.59 0.932 Upper limit of 95% CI 9.62 9.74 2.82 4.51 4.77 3.06 5.35 6.05 3.85 OR Quartile 4 31.4 16.4 2.49 3.79 3.93 2.44 4.55 4.91 2.81 p Value 8.3E−4 1.9E−4 0.019 0.0018 0.0013 0.022 3.9E−4 2.0E−4 0.0089 Lower limit of 95% CI 4.17 3.78 1.16 1.64 1.70 1.14 1.97 2.12 1.30 Upper limit of 95% CI 237 71.7 5.35 8.74 9.07 5.26 10.5 11.4 6.09

Example 20. Use of Insulin-Like Growth Factor-Binding Protein 6 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 6 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0), risk of injury (R), or injury (I) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 20.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 44.2 171 48.9 221 58.3 196 Average 163 507 168 559 197 542 Stdev 336 743 330 792 370 827 p (t-test) 1.5E−4 3.5E−5 6.3E−4 Min 0.259 3.12 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 98 51 106 43 113 36 sCr only Median 47.5 149 54.2 305 58.8 196 Average 164 497 166 562 198 521 Stdev 337 738 330 791 372 810 p (t-test) 2.3E−4 2.8E−5 0.0011 Min 0.259 3.12 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 97 52 106 43 111 38 UO only Median 62.9 263 63.7 305 63.7 305 Average 245 556 244 582 244 582 Stdev 482 799 481 815 481 815 p (t-test) 0.021 0.014 0.014 Min 0.259 12.3 0.259 12.3 0.259 12.3 Max 3600 3210 3600 3210 3600 3210 n (Patient) 130 18 131 17 131 17 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.71 0.72 0.73 0.72 0.73 0.70 0.68 0.73 SE 0.046 0.046 0.071 0.048 0.049 0.073 0.053 0.053 0.073 p Value 9.8E−7 4.3E−6 0.0018 2.3E−6 5.2E−6 0.0017 1.6E−4 4.8E−4 0.0017 nCohort Non-persistent 98 97 130 106 106 131 113 111 131 nCohort Persistent 51 52 18 43 43 17 36 38 17 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 88% 87% 94% 88% 86% 94% 86% 84% 94% Specificity 32% 31% 28% 30% 29% 27% 28% 28% 27% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 71% 69% 83% 72% 70% 82% 72% 68% 82% Specificity 60% 60% 55% 58% 58% 54% 57% 56% 54% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 45% 44% 44% 47% 49% 47% 44% 45% 47% Specificity 85% 85% 78% 83% 84% 78% 81% 81% 78% OR Quartile 2 3.47 2.88 6.51 3.29 2.55 6.06 2.45 2.07 6.06 p Value 0.010 0.022 0.074 0.022 0.056 0.086 0.088 0.14 0.086 Lower limit of 95% CI 1.34 1.16 0.836 1.18 0.977 0.776 0.875 0.787 0.776 Upper limit of 95% CI 8.99 7.12 50.7 9.12 6.65 47.4 6.86 5.43 47.4 OR Quartile 3 3.63 3.35 6.02 3.64 3.13 5.52 3.40 2.74 5.52 p Value 5.0E−4 9.3E−4 0.0063 0.0010 0.0031 0.0096 0.0034 0.011 0.0096 Lower limit of 95% CI 1.76 1.64 1.66 1.68 1.47 1.51 1.50 1.26 1.51 Upper limit of 95% CI 7.50 6.84 21.8 7.86 6.66 20.1 7.70 5.98 20.1 OR Quartile 4 4.55 4.34 2.79 4.25 5.00 3.13 3.31 3.47 3.13 p Value 1.4E−4 2.1E−4 0.048 3.0E−4 6.8E−5 0.031 0.0036 0.0022 0.031 Lower limit of 95% CI 2.09 2.00 1.01 1.94 2.26 1.11 1.48 1.56 1.11 Upper limit of 95% CI 9.90 9.42 7.71 9.32 11.0 8.83 7.41 7.69 8.83

TABLE 20.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 39.7 144 39.3 171 47.5 144 Average 164 463 163 506 197 479 Stdev 347 707 338 741 379 764 p (t-test) 7.8E−4 1.6E−4 0.0031 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 91 58 98 51 105 44 sCr only Median 40.8 144 39.7 196 50.3 144 Average 165 462 162 501 198 465 Stdev 347 708 339 735 382 750 p (t-test) 8.4E−4 1.7E−4 0.0045 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 91 58 97 52 103 46 UO only Median 59.0 119 59.4 123 59.4 123 Average 249 446 248 460 248 460 Stdev 493 707 491 718 491 718 p (t-test) 0.095 0.077 0.077 Min 0.259 5.59 0.259 5.59 0.259 5.59 Max 3600 3210 3600 3210 3600 3210 n (Patient) 123 25 124 24 124 24 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.70 0.67 0.73 0.73 0.68 0.70 0.69 0.68 SE 0.045 0.045 0.063 0.045 0.045 0.064 0.050 0.049 0.064 p Value 2.7E−6 8.9E−6 0.0057 2.8E−7 4.5E−7 0.0061 6.8E−5 1.5E−4 0.0061 nCohort Non-persistent 91 91 123 98 97 124 105 103 124 nCohort Persistent 58 58 25 51 52 24 44 46 24 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 86% 84% 92% 88% 87% 92% 86% 85% 92% Specificity 32% 31% 28% 32% 31% 28% 30% 29% 28% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 69% 69% 76% 73% 73% 75% 73% 72% 75% Specificity 62% 62% 55% 61% 62% 55% 59% 59% 55% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 41% 41% 36% 43% 44% 38% 41% 41% 38% Specificity 85% 85% 77% 84% 85% 77% 81% 82% 77% OR Quartile 2 2.92 2.42 4.57 3.47 2.88 4.33 2.65 2.29 4.33 p Value 0.015 0.039 0.047 0.010 0.022 0.056 0.046 0.074 0.056 Lower limit of 95% CI 1.23 1.05 1.02 1.34 1.16 0.966 1.02 0.922 0.966 Upper limit of 95% CI 6.96 5.60 20.4 8.99 7.12 19.4 6.91 5.69 19.4 OR Quartile 3 3.56 3.56 3.92 4.17 4.40 3.64 3.84 3.69 3.64 p Value 3.7E−4 3.7E−4 0.0066 1.5E−4 8.1E−5 0.010 6.0E−4 6.8E−4 0.010 Lower limit of 95% CI 1.77 1.77 1.46 2.00 2.11 1.35 1.78 1.74 1.35 Upper limit of 95% CI 7.15 7.15 10.5 8.72 9.20 9.80 8.30 7.83 9.80 OR Quartile 4 3.88 3.88 1.91 3.89 4.34 2.06 2.94 3.11 2.06 p Value 5.8E−4 5.8E−4 0.17 5.5E−4 2.1E−4 0.13 0.0063 0.0038 0.13 Lower limit of 95% CI 1.79 1.79 0.761 1.80 2.00 0.814 1.36 1.44 0.814 Upper limit of 95% CI 8.41 8.41 4.78 8.40 9.42 5.20 6.38 6.72 5.20

TABLE 20.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 39.3 119 38.5 161 47.4 119 Average 167 436 161 463 190 455 Stdev 355 685 348 700 366 737 p (t-test) 0.0021 6.4E−4 0.0038 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 86 63 90 59 98 51 sCr only Median 39.7 119 38.5 161 47.5 118 Average 168 430 161 463 191 447 Stdev 357 682 348 701 368 732 p (t-test) 0.0029 6.6E−4 0.0052 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 85 64 90 59 97 52 UO only Median 59.4 113 59.7 117 59.7 117 Average 244 422 243 432 243 432 Stdev 490 672 488 680 488 680 p (t-test) 0.097 0.081 0.081 Min 0.259 5.59 0.259 5.59 0.259 5.59 Max 3600 3210 3600 3210 3600 3210 n (Patient) 116 32 117 31 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.70 0.69 0.65 0.73 0.72 0.65 0.69 0.68 0.65 SE 0.044 0.044 0.058 0.044 0.044 0.058 0.048 0.048 0.058 p Value 4.7E−6 1.6E−5 0.0080 2.0E−7 4.1E−7 0.0089 9.9E−5 2.3E−4 0.0089 nCohort Non-persistent 86 85 116 90 90 117 98 97 117 nCohort Persistent 63 64 32 59 59 31 51 52 31 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 87% 86% 94% 90% 88% 94% 88% 87% 94% Specificity 34% 33% 30% 34% 33% 30% 32% 31% 30% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 67% 66% 69% 69% 69% 68% 69% 67% 68% Specificity 62% 61% 55% 62% 62% 55% 59% 59% 55% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 40% 39% 34% 42% 42% 35% 39% 38% 35% Specificity 85% 85% 78% 86% 86% 78% 82% 81% 78% OR Quartile 2 3.50 3.00 6.48 4.64 3.71 6.19 3.47 2.88 6.19 p Value 0.0046 0.010 0.014 0.0015 0.0044 0.016 0.010 0.022 0.016 Lower limit of 95% CI 1.47 1.30 1.47 1.80 1.51 1.40 1.34 1.16 1.40 Upper limit of 95% CI 8.32 6.94 28.6 12.0 9.16 27.4 8.99 7.12 27.4 OR Quartile 3 3.21 3.01 2.71 3.75 3.75 2.54 3.17 2.93 2.54 p Value 7.8E−4 0.0014 0.019 2.1E−4 2.1E−4 0.029 0.0016 0.0028 0.029 Lower limit of 95% CI 1.63 1.53 1.18 1.86 1.86 1.10 1.55 1.45 1.10 Upper limit of 95% CI 6.34 5.91 6.22 7.55 7.55 5.85 6.49 5.95 5.85 OR Quartile 4 3.69 3.55 1.81 4.36 4.36 1.92 2.87 2.74 1.92 p Value 9.7E−4 0.0014 0.17 2.3E−4 2.3E−4 0.13 0.0066 0.0091 0.13 Lower limit of 95% CI 1.70 1.64 0.775 1.99 1.99 0.819 1.34 1.29 0.819 Upper limit of 95% CI 8.03 7.71 4.24 9.52 9.52 4.53 6.13 5.85 4.53

TABLE 20.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 39.0 123 38.5 161 44.2 119 Average 162 438 161 463 182 460 Stdev 355 680 348 700 358 730 p (t-test) 0.0017 6.4E−4 0.0022 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 85 64 90 59 96 53 sCr only Median 39.7 119 39.0 144 47.5 118 Average 168 430 163 455 184 451 Stdev 357 682 350 697 359 726 p (t-test) 0.0029 9.2E−4 0.0031 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 85 64 89 60 95 54 UO only Median 58.5 118 58.9 118 58.9 118 Average 214 497 216 507 216 507 Stdev 382 829 380 851 380 851 p (t-test) 0.0056 0.0051 0.0051 Min 0.259 5.59 0.259 5.59 0.259 5.59 Max 2160 3600 2160 3600 2160 3600 n (Patient) 112 36 114 34 114 34 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.71 0.69 0.68 0.73 0.72 0.67 0.69 0.68 0.67 SE 0.043 0.044 0.054 0.044 0.044 0.056 0.047 0.047 0.056 p Value 1.2E−6 1.6E−5 0.0010 2.0E−7 9.3E−7 0.0026 4.2E−5 1.3E−4 0.0026 nCohort Non-persistent 85 85 112 90 89 114 96 95 114 nCohort Persistent 64 64 36 59 60 34 53 54 34 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 88% 86% 94% 90% 88% 94% 89% 87% 94% Specificity 34% 33% 31% 34% 34% 31% 32% 32% 31% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 67% 66% 69% 69% 68% 68% 68% 67% 68% Specificity 62% 61% 56% 62% 62% 55% 59% 59% 55% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 41% 39% 36% 42% 42% 35% 40% 39% 35% Specificity 86% 85% 79% 86% 85% 78% 82% 82% 78% OR Quartile 2 3.62 3.00 7.73 4.64 3.85 7.09 3.74 3.10 7.09 p Value 0.0036 0.010 0.0068 0.0015 0.0034 0.0096 0.0066 0.014 0.0096 Lower limit of 95% CI 1.52 1.30 1.76 1.80 1.56 1.61 1.44 1.25 1.61 Upper limit of 95% CI 8.62 6.94 34.0 12.0 9.49 31.2 9.67 7.65 31.2 OR Quartile 3 3.39 3.01 2.92 3.75 3.49 2.58 3.10 2.87 2.58 p Value 4.5E−4 0.0014 0.0087 2.1E−4 4.0E−4 0.021 0.0017 0.0031 0.021 Lower limit of 95% CI 1.72 1.53 1.31 1.86 1.75 1.15 1.53 1.43 1.15 Upper limit of 95% CI 6.71 5.91 6.51 7.55 6.97 5.79 6.27 5.77 5.79 OR Quartile 4 4.16 3.55 2.07 4.36 4.18 1.94 3.05 2.92 1.94 p Value 3.9E−4 0.0014 0.080 2.3E−4 3.3E−4 0.12 0.0040 0.0056 0.12 Lower limit of 95% CI 1.89 1.64 0.916 1.99 1.91 0.845 1.43 1.37 0.845 Upper limit of 95% CI 9.16 7.71 4.69 9.52 9.12 4.46 6.52 6.23 4.46

TABLE 20.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 37.9 126 37.5 171 40.8 123 Average 141 451 137 477 178 451 Stdev 309 688 302 702 361 713 p (t-test) 3.6E−4 9.3E−5 0.0023 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 82 67 86 63 93 56 sCr only Median 39.0 123 38.1 166 40.8 123 Average 146 450 141 476 164 474 Stdev 310 693 303 707 319 736 p (t-test) 4.8E−4 1.3E−4 5.1E−4 Min 0.259 0.723 0.259 3.12 0.259 3.12 Max 2160 3600 2160 3600 2160 3600 n (Patient) 83 66 87 62 93 56 UO only Median 58.3 119 58.5 123 58.5 123 Average 201 528 200 539 200 539 Stdev 358 839 357 848 357 848 p (t-test) 0.0012 8.2E−4 8.2E−4 Min 0.259 5.59 0.259 5.59 0.259 5.59 Max 2160 3600 2160 3600 2160 3600 n (Patient) 111 37 112 36 112 36 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.72 0.71 0.69 0.74 0.73 0.69 0.70 0.70 0.69 SE 0.043 0.043 0.053 0.042 0.043 0.054 0.046 0.046 0.054 p Value 2.5E−7 1.1E−6 3.1E−4 6.8E−9 4.0E−8 3.6E−4 2.1E−5 1.4E−5 3.6E−4 nCohort Non-persistent 82 83 111 86 87 112 93 93 112 nCohort Persistent 67 66 37 63 62 36 56 56 36 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 87% 86% 95% 89% 89% 94% 88% 88% 94% Specificity 34% 34% 32% 35% 34% 31% 32% 32% 31% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 67% 67% 70% 70% 69% 69% 68% 68% 69% Specificity 63% 63% 57% 64% 63% 56% 60% 60% 56% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 42% 41% 38% 44% 44% 39% 41% 41% 39% Specificity 88% 87% 79% 88% 87% 79% 84% 84% 79% OR Quartile 2 3.34 3.22 8.06 4.29 4.14 7.73 3.33 3.33 7.73 p Value 0.0048 0.0061 0.0057 0.0016 0.0020 0.0068 0.0090 0.0090 0.0068 Lower limit of 95% CI 1.45 1.40 1.83 1.74 1.68 1.76 1.35 1.35 1.76 Upper limit of 95% CI 7.72 7.45 35.4 10.6 10.2 34.0 8.23 8.23 34.0 OR Quartile 3 3.55 3.35 3.10 4.11 3.89 2.92 3.20 3.20 2.92 p Value 2.6E−4 4.7E−4 0.0055 6.8E−5 1.2E−4 0.0087 0.0011 0.0011 0.0087 Lower limit of 95% CI 1.80 1.70 1.40 2.05 1.94 1.31 1.59 1.59 1.31 Upper limit of 95% CI 7.00 6.61 6.90 8.23 7.79 6.51 6.42 6.42 6.51 OR Quartile 4 5.17 4.53 2.33 6.08 5.33 2.46 3.62 3.62 2.46 p Value 8.7E−5 2.2E−4 0.040 1.8E−5 4.9E−5 0.030 0.0010 0.0010 0.030 Lower limit of 95% CI 2.28 2.03 1.04 2.66 2.38 1.09 1.68 1.68 1.09 Upper limit of 95% CI 11.7 10.1 5.22 13.9 11.9 5.55 7.81 7.81 5.55

Example 21. Use of Insulin-Like Growth Factor-Binding Protein 6 for Evaluating Renal Status in Patients Admitted to the ICU: Persistent at RIFLE I or F

Patients from the intensive care unit (ICU) with RIFLE stage of injury (I) or failure (F) are enrolled in the following study. EDTA anti-coagulated blood samples (10 mL) and urine samples (50 mL) are collected from each patient at enrollment, and at every 12 hours up to day 3, and then every 24 hours thereafter up to day 7 while the subject is hospitalized. Insulin-like growth factor-binding protein 6 is measured in the enrollment samples by standard immunoassay methods using commercially available assay reagents.

Kidney status is assessed by RIFLE criteria based on serum creatinine, urine output, or both serum creatinine and urine output. Two cohorts are defined to represent a “persistent” and a “non-persistent” population. “Persistent” indicates those patients whose minimum RIFLE stage during a period of 24, 48 or 72 hours is injury (I) or failure (F) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. “Non-persistent” indicates those patients who are not persistent at injury (I) or failure (F) and whose minimum RIFLE stage during a period of 24, 48 or 72 hours is non-injury (RIFLE 0) or risk of injury (R) where the persistence period can start from the time of sample collection to 24, 48, 72, 96 or 168 hours after sample collection. If a patient dies after injury (I) or failure (F) or is placed on renal replacement therapy (RRT) at any time from sample collection to 24, 48, 72, 96 or 168 hours after sample collection, the patient is considered “persistent”.

The ability to distinguish the “persistent” and “non-persistent” cohorts is determined using receiver operating characteristic (ROC) analysis.

TABLE 21.1 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 24 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 22.2 118 37.9 117 39.3 171 Average 114 378 149 425 141 482 Stdev 312 612 315 676 299 713 p (t-test) 0.0033 0.0015 9.5E−5 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 55 94 78 71 88 61 sCr only Median 30.9 110 35.3 117 39.0 180 Average 124 378 153 413 146 480 Stdev 311 618 322 669 305 717 p (t-test) 0.0045 0.0028 1.4E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 57 92 76 73 89 60 UO only Median 59.4 138 63.7 190 63.7 243 Average 242 417 241 469 236 535 Stdev 495 649 483 713 476 755 p (t-test) 0.097 0.045 0.014 Min 0.259 2.91 0.259 7.04 0.259 7.04 Max 3600 3210 3600 3210 3600 3210 n (Patient) 114 34 121 27 125 23 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.71 0.66 0.69 0.69 0.67 0.72 0.72 0.70 SE 0.040 0.042 0.056 0.044 0.043 0.061 0.044 0.044 0.065 p Value 7.5E−10 3.7E−7 0.0032 1.6E−5 1.5E−5 0.0046 4.7E−7 4.3E−7 0.0021 nCohort Non-persistent 55 57 114 78 76 121 88 89 125 nCohort Persistent 94 92 34 71 73 27 61 60 23 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 85% 84% 91% 85% 85% 93% 85% 85% 91% Specificity 42% 39% 30% 33% 34% 29% 32% 31% 28% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 65% 63% 68% 63% 63% 70% 69% 68% 74% Specificity 75% 70% 55% 62% 62% 55% 62% 62% 54% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 36% 36% 35% 38% 37% 37% 43% 43% 43% Specificity 93% 91% 78% 86% 86% 78% 86% 87% 78% OR Quartile 2 4.11 3.23 4.39 2.73 2.93 5.09 2.70 2.60 4.08 p Value 3.9E−4 0.0028 0.020 0.014 0.0082 0.033 0.020 0.025 0.066 Lower limit of 95% CI 1.88 1.50 1.26 1.23 1.32 1.14 1.17 1.13 0.909 Upper limit of 95% CI 8.97 6.96 15.3 6.05 6.51 22.6 6.23 6.01 18.3 OR Quartile 3 5.41 4.01 2.58 2.77 2.76 2.85 3.68 3.49 3.38 p Value 7.7E−6 1.2E−4 0.021 0.0027 0.0027 0.023 2.2E−4 4.0E−4 0.016 Lower limit of 95% CI 2.58 1.98 1.15 1.43 1.42 1.16 1.84 1.75 1.25 Upper limit of 95% CI 11.3 8.15 5.79 5.38 5.36 7.01 7.37 6.97 9.14 OR Quartile 4 7.22 5.82 1.94 3.74 3.47 2.05 4.70 4.91 2.79 p Value 4.3E−4 6.5E−4 0.12 0.0012 0.0022 0.11 1.3E−4 8.7E−5 0.030 Lower limit of 95% CI 2.40 2.11 0.845 1.68 1.56 0.840 2.13 2.22 1.10 Upper limit of 95% CI 21.7 16.0 4.46 8.30 7.69 4.99 10.4 10.9 7.06

TABLE 21.2 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 48 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 21.6 117 33.8 118 37.5 161 Average 115 375 148 408 140 453 Stdev 315 610 324 657 308 687 p (t-test) 0.0041 0.0027 3.0E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 54 95 73 76 82 67 sCr only Median 28.9 109 36.8 114 37.5 161 Average 125 374 155 401 142 450 Stdev 314 616 327 659 311 688 p (t-test) 0.0057 0.0046 4.0E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 56 93 73 76 82 67 UO only Median 58.2 124 58.8 126 58.8 221 Average 213 457 213 505 209 559 Stdev 390 775 382 837 376 876 p (t-test) 0.013 0.0046 0.0011 Min 0.259 2.91 0.259 5.59 0.259 5.59 Max 2160 3600 2160 3600 2160 3600 n (Patient) 106 42 113 35 117 31 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.71 0.67 0.70 0.68 0.68 0.73 0.72 0.70 SE 0.040 0.042 0.051 0.043 0.044 0.055 0.042 0.042 0.057 p Value 1.4E−9 6.2E−7 6.9E−4 2.9E−6 3.5E−5 0.0012 7.0E−8 1.9E−7 6.0E−4 nCohort Non-persistent 54 56 106 73 73 113 82 82 117 nCohort Persistent 95 93 42 76 76 35 67 67 31 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 85% 84% 90% 86% 84% 91% 87% 85% 90% Specificity 43% 39% 31% 36% 34% 30% 34% 33% 29% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 64% 62% 69% 63% 62% 71% 69% 69% 74% Specificity 74% 70% 58% 63% 62% 57% 65% 65% 56% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 36% 35% 36% 37% 36% 37% 40% 40% 42% Specificity 93% 91% 79% 86% 85% 79% 87% 87% 79% OR Quartile 2 4.29 3.36 4.29 3.27 2.78 4.59 3.34 2.80 3.82 p Value 2.6E−4 0.0020 0.010 0.0037 0.011 0.017 0.0048 0.013 0.036 Lower limit of 95% CI 1.96 1.56 1.42 1.47 1.27 1.32 1.45 1.24 1.09 Upper limit of 95% CI 9.39 7.27 13.0 7.27 6.08 16.0 7.72 6.32 13.4 OR Quartile 3 5.13 3.80 3.02 2.92 2.60 3.27 4.00 4.00 3.72 p Value 1.5E−5 2.2E−4 0.0043 0.0016 0.0045 0.0048 7.5E−5 7.5E−5 0.0036 Lower limit of 95% CI 2.45 1.87 1.42 1.50 1.35 1.43 2.01 2.01 1.54 Upper limit of 95% CI 10.7 7.71 6.46 5.68 5.04 7.43 7.95 7.95 9.00 OR Quartile 4 6.97 5.61 2.12 3.68 3.11 2.19 4.36 4.36 2.80 p Value 5.5E−4 8.4E−4 0.061 0.0017 0.0052 0.061 3.2E−4 3.2E−4 0.017 Lower limit of 95% CI 2.32 2.04 0.966 1.63 1.40 0.965 1.96 1.96 1.20 Upper limit of 95% CI 21.0 15.4 4.66 8.29 6.88 4.98 9.71 9.71 6.50

TABLE 21.3 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 72 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 21.6 117 33.4 117 35.3 126 Average 115 375 148 404 140 435 Stdev 315 610 327 654 315 672 p (t-test) 0.0041 0.0033 6.8E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 54 95 72 77 78 71 sCr only Median 28.9 109 35.3 111 37.5 119 Average 125 374 156 397 147 427 Stdev 314 616 329 655 318 674 p (t-test) 0.0057 0.0055 0.0012 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 56 93 72 77 78 71 UO only Median 58.5 118 58.9 118 58.9 123 Average 221 411 217 448 212 485 Stdev 400 735 391 778 384 809 p (t-test) 0.043 0.018 0.0065 Min 0.259 2.91 0.259 3.55 0.259 3.55 Max 2160 3600 2160 3600 2160 3600 n (Patient) 100 48 106 42 110 38 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.71 0.64 0.70 0.68 0.65 0.73 0.71 0.66 SE 0.040 0.042 0.050 0.042 0.043 0.052 0.042 0.043 0.054 p Value 1.4E−9 6.2E−7 0.0042 2.1E−6 2.6E−5 0.0038 6.0E−8 1.3E−6 0.0026 nCohort Non-persistent 54 56 100 72 72 106 78 78 110 nCohort Persistent 95 93 48 77 77 42 71 71 38 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 85% 84% 90% 86% 84% 90% 87% 86% 89% Specificity 43% 39% 32% 36% 35% 31% 36% 35% 30% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 64% 62% 65% 64% 62% 67% 68% 66% 68% Specificity 74% 70% 57% 64% 62% 57% 65% 64% 56% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 36% 35% 31% 36% 35% 33% 39% 38% 37% Specificity 93% 91% 78% 86% 85% 78% 87% 86% 79% OR Quartile 2 4.29 3.36 4.05 3.39 2.88 4.29 3.86 3.23 3.64 p Value 2.6E−4 0.0020 0.0071 0.0027 0.0082 0.010 0.0016 0.0048 0.023 Lower limit of 95% CI 1.96 1.56 1.46 1.53 1.32 1.42 1.67 1.43 1.20 Upper limit of 95% CI 9.39 7.27 11.2 7.54 6.31 13.0 8.92 7.30 11.1 OR Quartile 3 5.13 3.80 2.42 3.10 2.76 2.61 3.94 3.50 2.80 p Value 1.5E−5 2.2E−4 0.015 9.2E−4 0.0027 0.012 8.0E−5 2.8E−4 0.0098 Lower limit of 95% CI 2.45 1.87 1.19 1.59 1.42 1.24 1.99 1.78 1.28 Upper limit of 95% CI 10.7 7.71 4.93 6.04 5.36 5.51 7.79 6.87 6.11 OR Quartile 4 6.97 5.61 1.61 3.54 2.99 1.80 4.43 3.74 2.21 p Value 5.5E−4 8.4E−4 0.23 0.0023 0.0068 0.14 3.6E−4 0.0012 0.054 Lower limit of 95% CI 2.32 2.04 0.744 1.57 1.35 0.818 1.96 1.68 0.988 Upper limit of 95% CI 21.0 15.4 3.49 7.99 6.63 3.98 10.0 8.30 4.93

TABLE 21.4 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 96 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 21.6 117 33.4 117 35.3 126 Average 115 375 148 404 140 435 Stdev 315 610 327 654 315 672 p (t-test) 0.0041 0.0033 6.8E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 54 95 72 77 78 71 sCr only Median 28.9 109 35.3 111 37.5 119 Average 125 374 156 397 147 427 Stdev 314 616 329 655 318 674 p (t-test) 0.0057 0.0055 0.0012 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 56 93 72 77 78 71 UO only Median 58.5 118 58.8 117 58.8 119 Average 223 399 220 424 216 457 Stdev 403 723 396 756 389 785 p (t-test) 0.060 0.033 0.014 Min 0.259 2.91 0.259 3.55 0.259 3.55 Max 2160 3600 2160 3600 2160 3600 n (Patient) 98 50 103 45 107 41 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.74 0.71 0.64 0.70 0.68 0.65 0.73 0.71 0.66 SE 0.040 0.042 0.049 0.042 0.043 0.051 0.042 0.043 0.052 p Value 1.4E−9 6.2E−7 0.0040 2.1E−6 2.6E−5 0.0041 6.0E−8 1.3E−6 0.0030 nCohort Non-persistent 54 56 98 72 72 103 78 78 107 nCohort Persistent 95 93 50 77 77 45 71 71 41 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 85% 84% 90% 86% 84% 91% 87% 86% 90% Specificity 43% 39% 33% 36% 35% 32% 36% 35% 31% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 64% 62% 64% 64% 62% 64% 68% 66% 66% Specificity 74% 70% 57% 64% 62% 56% 65% 64% 56% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 36% 35% 30% 36% 35% 31% 39% 38% 34% Specificity 93% 91% 78% 86% 85% 78% 87% 86% 79% OR Quartile 2 4.29 3.36 4.36 3.39 2.88 4.83 3.86 3.23 4.12 p Value 2.6E−4 0.0020 0.0045 0.0027 0.0082 0.0053 0.0016 0.0048 0.012 Lower limit of 95% CI 1.96 1.56 1.58 1.53 1.32 1.60 1.67 1.43 1.36 Upper limit of 95% CI 9.39 7.27 12.1 7.54 6.31 14.6 8.92 7.30 12.5 OR Quartile 3 5.13 3.80 2.37 3.10 2.76 2.34 3.94 3.50 2.46 p Value 1.5E−5 2.2E−4 0.016 9.2E−4 0.0027 0.022 8.0E−5 2.8E−4 0.019 Lower limit of 95% CI 2.45 1.87 1.17 1.59 1.42 1.13 1.99 1.78 1.16 Upper limit of 95% CI 10.7 7.71 4.79 6.04 5.36 4.82 7.79 6.87 5.21 OR Quartile 4 6.97 5.61 1.48 3.54 2.99 1.57 4.43 3.74 1.89 p Value 5.5E−4 8.4E−4 0.32 0.0023 0.0068 0.26 3.6E−4 0.0012 0.11 Lower limit of 95% CI 2.32 2.04 0.686 1.57 1.35 0.718 1.96 1.68 0.857 Upper limit of 95% CI 21.0 15.4 3.19 7.99 6.63 3.44 10.0 8.30 4.19

TABLE 21.5 Comparison of marker levels and the area under the ROC curve (AUC) in urine samples for the “persistent” and “non-persistent” cohorts where persistence starts within 168 hours after sample collection and renal status is assessed by serum creatinine (sCr) only, urine output (UO) only, or serum creatinine or urine output RIFLE criteria. Persistence Period Duration (hr) 24 48 72 Non-persistent Persistent Non-persistent Persistent Non-persistent Persistent Cohort Cohort Cohort Cohort Cohort Cohort sCr or UO Median 21.6 117 32.9 118 35.3 126 Average 107 374 147 396 141 419 Stdev 311 605 332 643 322 658 p (t-test) 0.0034 0.0044 0.0014 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 52 97 69 80 74 75 sCr only Median 26.8 110 33.4 117 35.3 126 Average 115 377 145 400 137 430 Stdev 307 614 327 647 315 665 p (t-test) 0.0037 0.0034 7.3E−4 Min 0.259 0.723 0.259 0.723 0.259 0.723 Max 2160 3600 2160 3600 2160 3600 n (Patient) 55 94 70 79 76 73 UO only Median 54.3 121 58.3 119 54.3 138 Average 207 422 207 434 203 459 Stdev 380 729 376 749 371 767 p (t-test) 0.019 0.015 0.0070 Min 0.259 2.91 0.259 2.91 0.259 2.91 Max 2160 3600 2160 3600 2160 3600 n (Patient) 96 52 99 49 102 46 Persistence Period Duration (hr) 24 48 72 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.75 0.72 0.66 0.70 0.70 0.65 0.72 0.72 0.67 SE 0.040 0.041 0.048 0.042 0.042 0.049 0.042 0.042 0.050 p Value 4.5E−10 7.2E−8 8.4E−4 1.2E−6 2.2E−6 0.0018 2.3E−7 6.8E−8 8.9E−4 nCohort Non-persistent 52 55 96 69 70 99 74 76 102 nCohort Persistent 97 94 52 80 79 49 75 73 46 Cutoff Quartile 2 17.2 17.2 17.6 17.2 17.2 17.6 17.2 17.2 17.6 Sensitivity 85% 84% 90% 85% 85% 90% 85% 86% 89% Specificity 42% 40% 33% 36% 36% 32% 35% 36% 31% Cutoff Quartile 3 78.3 78.3 80.9 78.3 78.3 80.9 78.3 78.3 80.9 Sensitivity 64% 63% 65% 64% 63% 65% 67% 67% 67% Specificity 75% 71% 58% 65% 64% 58% 66% 66% 58% Cutoff Quartile 4 331 331 331 331 331 331 331 331 331 Sensitivity 36% 36% 33% 36% 37% 33% 39% 40% 35% Specificity 94% 93% 79% 87% 87% 79% 88% 88% 79% OR Quartile 2 4.01 3.51 4.70 3.22 3.10 4.20 3.15 3.47 3.75 p Value 4.7E−4 0.0014 0.0028 0.0035 0.0047 0.0056 0.0048 0.0028 0.011 Lower limit of 95% CI 1.84 1.62 1.70 1.47 1.41 1.52 1.42 1.54 1.35 Upper limit of 95% CI 8.73 7.60 13.0 7.07 6.80 11.6 7.00 7.85 10.4 OR Quartile 3 5.31 4.11 2.64 3.30 3.10 2.55 3.92 3.93 2.84 p Value 1.3E−5 1.1E−4 0.0065 5.1E−4 9.2E−4 0.0097 8.3E−5 8.2E−5 0.0052 Lower limit of 95% CI 2.50 2.01 1.31 1.68 1.59 1.26 1.99 1.99 1.37 Upper limit of 95% CI 11.3 8.41 5.33 6.46 6.06 5.20 7.74 7.75 5.89 OR Quartile 4 9.22 7.22 1.85 3.79 3.93 1.80 4.55 4.91 2.06 p Value 4.3E−4 4.3E−4 0.11 0.0018 0.0013 0.13 3.9E−4 2.0E−4 0.068 Lower limit of 95% CI 2.68 2.40 0.863 1.64 1.70 0.836 1.97 2.12 0.949 Upper limit of 95% CI 31.8 21.7 3.95 8.74 9.07 3.88 10.5 11.4 4.46

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims. 

1-111. (canceled)
 112. A method of treating acute kidney injury comprising: (a) detecting a level of one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6 in a body fluid sample obtained from a subject; (b) determining that the subject is at increased risk of persistence of a RIFLE I acute kidney injury or persistence of a RIFLE F acute kidney injury based on the level of one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6 in the body fluid sample; and (c) administering to the subject at the increased risk of persistence of a RIFLE I acute kidney injury or persistence of a RIFLE F acute kidney injury a treatment regimen comprising one or more of renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, monitoring and optimizing hemodynamics and fluid administration, and adjusting dosing of renally excreted substances.
 113. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of the persistence of a RIFLE I acute kidney injury or the persistence of a RIFLE F acute kidney injury for a period of 24 hours or more after the time the sample is obtained from the subject.
 114. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of the persistence of a RIFLE I acute kidney injury or the persistence of a RIFLE F acute kidney injury for a period of 48 hours or more after the time the sample is obtained from the subject.
 115. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of the persistence of a RIFLE I acute kidney injury or the persistence of a RIFLE F acute kidney injury for a period of 72 hours or more after the time the sample is obtained from the subject.
 116. The method of claim 112, wherein the determining comprises determining that the subject has an increased likelihood of the persistence of a RIFLE I acute kidney injury or the persistence of a RIFLE F acute kidney injury for a period of 24, 48, or 72 hours after the time the sample is obtained from the subject.
 117. The method of claim 112, wherein the persistence of a RIFLE I acute kidney injury or the persistence of a RIFLE F acute kidney injury is future persistence of a RIFLE I acute kidney injury or future persistence of a RIFLE F acute kidney injury.
 118. The method of claim 112, wherein the persistence of a RIFLE I acute kidney injury or the persistence of a RIFLE F acute kidney injury is future persistence of a RIFLE F acute kidney injury.
 119. The method of claim 112, wherein the persistence of a RIFLE I acute kidney injury or the persistence of a RIFLE F acute kidney injury is future persistence of a RIFLE I acute kidney injury.
 120. The method of claim 117, wherein the future persistence comprises a persistence period that starts within 24 hours after the sample is obtained.
 121. The method of claim 117, wherein the future persistence comprises a persistence period that starts within 48 hours after the sample is obtained.
 122. The method of claim 117, wherein the future persistence comprises a persistence period that starts within 72 hours after the sample is obtained.
 123. The method of claim 112, wherein the persistence comprises a persistence period that starts at the time the sample is obtained.
 124. The method of claim 112, wherein the detecting comprises detecting an elevated level of the one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6 in the sample as compared to a predetermined threshold level.
 125. The method of claim 112, wherein the detecting comprises: contacting all or a portion of the sample with a binding reagent which binds to the one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6; and generating an assay result indicative of binding of the one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein
 6. 126. The method of claim 125, wherein the assay result is a measured concentration of the one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein
 6. 127. The method of claim 125, wherein the assay result is a composite of a plurality of assay results.
 128. The method of claim 125, wherein the binding reagent is an antibody.
 129. The method of claim 112, wherein the body fluid sample is a urine sample.
 130. A method of treating acute kidney injury comprising: (a) detecting a level of one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6 in a urine sample obtained from a subject; (b) determining that the subject is at increased risk of persistence of a RIFLE F acute kidney injury based on the level of one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6 in the sample, wherein the persistence comprises a persistence period that starts at or after the time the sample is obtained and at or before 48 hours after the sample is obtained, and lasts for a period of 72 hours or more; and (c) administering to the subject at the increased risk of persistence of a RIFLE F acute kidney injury a treatment regimen comprising one or more of renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, monitoring and optimizing hemodynamics and fluid administration, and adjusting dosing of renally excreted substances.
 131. A method of treating acute kidney injury comprising: (a) detecting a level of one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6 in a urine sample obtained from a subject; (b) determining that the subject is at increased risk of persistence of a RIFLE I acute kidney injury based on the level of one or more of insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, insulin-like growth factor-binding protein 4, and insulin-like growth factor-binding protein 6 in the sample, wherein the persistence comprises a persistence period that starts at or after the time the sample is obtained and at or before 48 hours after the sample is obtained, and lasts for a period of 72 hours or more; and (c) administering to the subject at the increased risk of persistence of a RIFLE I acute kidney injury a treatment regimen comprising one or more of renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, monitoring and optimizing hemodynamics and fluid administration, and adjusting dosing of renally excreted substances. 